Method for the identification of target epitopes of the t-cell mediated immune response and for the detection of epitope specific cells

ABSTRACT

The present invention relates to a method for the detection of epitope-specific T-cells and target epitopes of reactive T-cells. Furthermore, the present invention relates to vectors comprising a first promoter which is specifically inducible by the epitope-specific contact with a T-cell in antigen-presenting cells, a nucleic acid which is functionally linked to this first promoter and which encodes a marker gene, a second promoter which is constitutive in antigen-presenting cells, and a nucleic acid which is functionally linked to said second promoter. Furthermore, the present invention relates to antigen-presenting cells which are transduced with the vectors according to the invention.

The present invention relates to a method for the detection ofepitope-specific T-cells and target epitopes of reactive T-cells.Furthermore, the present invention relates to vectors comprising a firstpromoter which is specifically inducible by the epitope-specific contactwith a T-cell in antigen-presenting cells, a nucleic acid which isfunctionally linked to this first promoter and which encodes a markergene, a second promoter which is constitutive in antigen-presentingcells, and a nucleic acid which is functionally linked to said secondpromoter. Furthermore, the present invention relates toantigen-presenting cells which are transduced with the vectors accordingto the invention.

The acquired branch of the immune system consists of a humoral(immunoglobulins) and a cellular immune defence. Cellular andpathogen-specific polypeptides are processed by cells by specificcleavage and fragments (epitopes) thereof are presented together withMHC-molecules of the class-I and/or -II on the surface ofantigen-presenting cells (APC). By means of their T-cell receptorT-cells specifically recognise epitopes which are presented in thecomplex with the body's own MHC-proteins and initiate an immunereaction.

T-cells can be partitioned into different effector populations on thebasis of specific surface proteins. CD4⁺ T-helper cells play a centralrole in the control of the immune defence. After a specific recognitionof epitopes being presented to them on the surface of APC together withMHC-proteins, they regulate the production of antibodies by B-cells(humoral branch of the immune response) by the secretion of differentmessenger substances (for example cytokines) and the activation of CD8⁺cytotoxic T-cells (CTL) (cellular branch of the immune response). Themeaning of CD8⁺ CTL resides in the recognition and the destruction ofdegenerated cells and tissues as well as cells and tissues which havebeen attacked by micro-organisms or parasites. T-cells therefore displaya prominent mechanism for protection of the acquired immune system forthe prevention and control of microbial, especially virus-contingentdiseases and for the recognition and destruction of degenerated cells ofthe body.

Professional APC such as dendritic cells, monocytes, macrophages, butalso non-professional APC such as B-cells play a central role both intriggering the T-cell-response against exogenous immunogens and also inthe induction of a T-cell-tolerance against tissues in the body itself.The activation and proliferation of T-cells is accomplished by thesimultaneous triggering of two signals. The first signal is transducedinto the T-cell by the T-cell receptor which recognises an epitope inconnection with MHC on the surface of APC. The second co-stimulatingsignal is mediated by the specific interaction of the co-stimulatingmolecules B7.1 (CD80) or B7.2 (CD86) on the APC with the cognatereceptor (CD28) on the surface of the T-cell. In the absence of theco-stimulating signal the T-cell becomes anergic. Anergy describes astate in which the T-cells do not proliferate and do not react to anantigen.

The degree of activation of an APC and the composition of a foreignsubstance significantly decide on the profile of the induced immuneresponse. Thus, the concentration and biochemical characteristics of aforeign substance as well as the presence or absence ofimmune-modulating substances (especially bacterial lipopolysaccharides(LPS), bacterial nucleic acids (with CpG-motives) and exogenouspolypeptides (e.g. bacterial flagellin)) have a significant effectwhether the cellular (Th-1 cell-mediated immunity) or the humoral branch(TH-2 cell-mediated immune response) of the immune system is activatedor whether the immune response comes up tolerogenically.

In order to prevent undesired immune reactions against proteins andtissues of the body itself, auto-reactive T-cells are early eliminated(clonal deletion) or inactivated (anergy). Consequence is anantigen-specific tolerance against structures (polypeptides, cells,tissues and organs) of the body itself. In the case of auto-immunediseases these protection mechanisms are disturbed or onlyinsufficiently developed. By now the causes forming the bases of theformation of said auto-immune diseases are only poorly understood.Possibly, structures of the body itself are wrongly recognised asforeign due to their similarity to polypeptides specific to pathogensand foreign tissue and damaged by misguided effectors of the acquiredimmune system (T-cells and/or antibodies) or even destroyed.

Besides this, in connection with chronically persisting viral infectionsit does not seldom amount to inflammatory processes, partly afflictingvital organs such as the liver (viral hepatides) although the immuneresponse is targeted primarily against the structures which arepathogen-specific. In such cases, it is spoken of immunopathogenesissince the damage and the symptoms of the disease are primarily caused bythe own immune system and not by the pathogen.

Similar immune reactions form the basis also of the rejection oftransplanted tissues and organs. In this case a combination of foreignMHC-proteins of the donor and epitopes of the body itself is recognisedas foreign by T-cells of the recipient and attacked.

In contrast to the above mentioned diseases, in the case of tumourdiseases often an insufficient immune recognition can be observed.Frequently tumour infiltrating lymphocytes (TIL) can be detectedattacking the tumour in a limited scope but having a specificity onmolecular level which is mostly not known. The reason why these TILs arenot able in vivo to eliminate the tumour is unclear. The concentrationof said TILs in the blood, usually being very low and often even underthe threshold of detection, is presumably a decisive factor. For thestimulation and enhancement of such a tumour-specific immune response itis tried nowadays to stimulate TILs with tumour material ex vivo(outside of the body) and to bring them back into the body. In the caseof other approaches tumour material from biopsies is labelled ex vivo bymeans of different antigens and brought back into the body afterirradiation. The idea is hereby to make the tumour notable for theimmune system by means of the new antigens and to stimulate thereby alsoTILs against naturally important tumour associated antigens by thedestruction of tumour cells in vivo. However, these approaches areeffective only in a very limited scope since the tumour cells can hardlystimulate an immune reaction. On the one hand, the reasons reside in thelow concentration of specific antigens. On the other hand, the lack ofco-stimulating molecules or MHC molecules leads to an inefficientpresentation.

Moreover, the cellular immune response plays a central role in thecontrol of numerous viral infects like for example HIV infections orherpes virus infections. Taking into consideration the importance of thecellular immune response in the control of microbial infects andtumours, at present manifold new immunisation strategies for theinduction of antigen-specific T-cells are being tested. These comprisethe application of live attenuated viruses and bacteria, recombinantlive vaccines (based on different recombinant bacteria and viruses),particulate immunogens, (lipo-) proteins, (lipo-) peptides andDNA-vaccines. In addition, different forms of administration of thesegroups of vaccines, for example the combination of different vaccines inthe “prime-boost” method and the combined administration with adjuvantsand carrier substances, are tested for their applicability for theinduction of a T-cell response. Most of the mentioned immunisationvectors are principally suited to elicit also a CD8⁺ T-cell-mediatedcytotoxic immune response besides a CD4⁺ T-helper cell response.However, the efficiency of these methods for the targeted induction ofan efficient CD8⁺ T-cell response is up to now limited by the fact thatin the case of very many diseases induced by micro-organisms and in thecase of tumours the target epitopes of the protective T-cells areunknown, or only known to a limited number. The targeted insertion ofrationally selected T-cell epitopes would lead to a significant increaseof effectivity and efficiency of the beforehand described immunisationvectors.

Particularly in the case of vaccines and immunisation vectors with alimited capacity for the insertion of foreign genes (particularlyDNA-vaccines, repticons, recombinant bacterial and viral vectors) orforeign epitopes (particularly vaccines based on peptides, polypeptides,lipoproteins and chimeric particulated immunogens) the knowledge ofrelevant target structures of the T-cell response is of decisiveimportance. In the case of the untargeted expression of a great numberof epitopes it can furthermore amount to a redirection of the immuneresponse to non-relevant targets because of (to a great extent unknown)epitopes.

By the presently available immunological techniques for theidentification of target epitopes of reactive T-cells the APC areusually changed in such a way, that they present previously selectedepitopes of polypeptides on their surface. The altered APC are herebyincubated together with autologous T-cells, or such cells thatcorrespond to the autologic T-cells in terms of the recognition ofMHC-molecules, and specific reactions of the T-cells (for exampleproliferation, cytokine release or cytotoxic activity) are measuredafter a specific recognition of epitopes/MHC complexes on the surface ofAPC.

For this, APC are for example incubated with peptides or mixtures ofdifferent peptides containing potential or already known target epitopesof reactive T-cells. In the case of this experimental approach thepeptides are directly loaded on membrane associated MHC-proteins.Alternatively, polynucleotides encoding polypeptides which containpotential target epitopes, or reactive T-cells can be introduced intoAPC by means of transduction of different recombinant transfer systems(plasmids, non-viral or viral vectors) and the polypeptides can beexpressed in the APC. These are then processed in the cytoplasm of theAPC, loaded on MHC proteins of the classes I and II and presentedtogether with them on the surface of the APC.

Alternatively, an epitope-loading of MHC proteins can be achieved by anincubation of APC with polypeptides, (lipo-) proteins or (lipo-) proteinaggregates. In order to increase the uptake of these polypeptides orpolypeptide-complexes, these can be subjected to a treatment withphysical (denaturation with heat) or chemical methods(sodium-dodecylsulfate (SDS), urea or acid treatment). Moreover, thefusion of the polypeptides with CpG-containing nucleic acids orpolypeptides of the SV40 T-antigen enhances their uptake, processing andepitope presentation. Polypeptides that potentially bear any antigenicepitopes are beforehand purified from organs, tissues, cells,micro-organisms or any other biological material by biochemical means.In addition, the polypeptides can be produced recombinantly for exampleby the means of genetically modified cells, yeasts, bacteria or virusesand be purified from them. Furthermore, also the incubation of APC withvital or killed bacteria, yeasts, mammalian and insect cells producingthe polypeptide, or with their lysates is suited for the introduction ofthese polypeptides into the MHC-class I and -II pathway of antigenprocessing and antigen presentation. Subsequently the in such a waypre-treated APC are co-cultivated with T-cells or T-cell-containing cellmixtures. The identification of polypeptides containing target epitopesof reactive T-cells is performed by the determination of characteristicreactions of reactive T-cells (for example proliferation, cytokinerelease or cytotoxic activity) due to a specific recognition ofepitope/MHC complexes on the surface of the APC. The like also appliesfor the determination of tumour associated antigens being recognised bytumour infiltrating lymphocytes.

As an alternative to single polypeptides which have been selectedbeforehand, theoretically also a great number of polypeptides can besimultaneously tested for their recognition by T-cells. For thispurpose, polynucleotides of a gene library are introduced into APC bymeans of different non-viral and viral transfer systems which forexample encode all expressed polypeptides of a cell. The modified APCare again incubated with T-cells and distributed over the wells ofmicro-titre-plates. The identification of target epitopes orpolypeptides containing target epitopes of reactive T-cells is againperformed by the determination of a specific reaction of the T-cells.Since mixtures of different APC presenting different epitopes areconcerned, the APC of one well showing T-cell recognition are againdistributed over different wells of a further micro-titre-plate(limiting dilution) and again tested for a recognition by specificT-cells. This dilution method is repeated until each well that shows ameasurable T-cell reaction statistically represents only derivatives ofone epitope presenting APC.

However, none of the methods for the search for epitopes which areavailable by now is suited to determine the target epitopes ofprotective or auto-aggressive T-cells in the scope of an acceptableeffort for costs and labour and at a speed and completeness which isrequired for the manifold, urgently needed prophylactic, therapeutic anddiagnostic applications. The use of the above mentioned methods for thequick and all-embracing determination of T-cell epitopes fails in thefirst instance because of the problems given below.

The peptide loading of membrane associated MHC proteins on APC indeedrepresents a very efficient method for stimulation of T-cells, but thereis the considerable restriction that only known peptide sequences can betested by this method. There is the same limitation also for methodswhich are based on the incubation of the APC with purified polypeptides,(lipo-) proteins, (lipo-) protein aggregates, cell lysates or apoptoticpolypeptide producing cells. Methods for transformation of APC withpolynucleotides encoding these polypeptides show the same restrictions.This restriction on the few known epitopes or polypeptides can not becircumvented by an increase of the number of polynucleotides,polypeptides or cell lysates tested due to the complexity of thebiological detection systems. So for example, the exact analysis of theT-cell recognition of a single protein with a medium size of 20-40 kDa(about 200 to 400 amino acids) requires the synthesis and examination ofmore than 50 overlapping peptides. According to recent estimates basedon the results of the human genome project for the enlightenment of thehuman genome, the total number of the human genes ad up to about 30000.The total number of proteins being specifically expressed in adifferentiated cell and which all may contain putative target epitopesof reactive T-cells is therefore clearly too high to be all-embracinglytested for the presence of T-cell epitopes by means of the molecularbiological and biochemical methods, respectively, available by now.

Screening methods of gene libraries for the search for T-cell epitopeswhich are available at present have also two decisive limitations. Dueto the very high time exposure of these analyses of several weeks anddue to the limited life span of the modified APC and the T-cells inculture, these analyses usually can not be performed with primary APCand T-cells because of the limited ability to perform cell divisions.Instead, these analyses require the generation of immortalised T-celland APC populations. In either case this step is very time consuming andbecause of the necessity of corresponding MHC patterns limited in itsapplication.

Due to the use of immortalised T-cell lines furthermore a reduction ofthe originally polyclonal T-cell population to oligoclonal T-cell linestakes place. Even in the case of the analysis of tumour infiltratinglymphocytes in which single T-cell clones are accumulated, these have tobe first sub-cloned for the purpose of analysis.

The use of arbitrarily selected peptides and polypeptides for the searchfor T-cells is therefore not feasible because of the high number ofpotential targets of a T-cell response and because of theMHC-restriction of the specific epitope recognition by T-cells as wellas the extremely high costliness and time exposure for the productionand purification of polypeptides and for the performance of thepresently available methods for the search of epitopes.

For the detection of T-cells with a certain antigen specificity in apopulation of T-cells, the T-cells are co-incubated with APC whichpresent specific epitopes. The detection of antigen-specific CD4⁺T-helper cells is subsequently performed either by the determination ofthe secretion of cytokine (by FACS, ELISPOT or ELISA technique), by themeasurement of the T-cell proliferation (by determination of theincorporation of ³H tritium into the DNA of proliferating cells) afterspecific re-stimulation of the T-cells with polypeptides or epitopeswith a suitable length or by the detection of epitope-specific T-cellsby means of the dimer or tetramer technology.

The detection of specific CD8⁺ T-cells (CTL) is performed by aco-cultivation of these cells with peptide loaded APC and thedetermination of the cytolytic activity of the CD8⁺ CTL (for example bymeans of the ⁵¹chromium release assay) or by the measurement of theIFN-γ secretion from the CTL (by FACS, ELISPOT or ELISA technique).

Alternatively, epitope-specific T-cells can be again specificallydetected by means of the dimer or tetramer technology.

One of the disadvantages of the presently available methods for thedetection of T-cells with a certain antigen-specificity resides in theirlow sensitivity. Up to now, it is only possible to measure the reactionof the T-cells as a result of an epitope-specific recognition of theAPC. Thus, the sensitivity of the detection is directly dependent on theconcentration of the epitope-specific T-cells. In the case of thescreening of gene libraries this forms a particular disadvantage. Due tothe relatively high number of polynucleotide fragments in a gene librarythe number of copies of the single polynucleotide fragments of the genelibrary is very low after the introduction into the APC.

A pre-stimulation of the T-cell population with epitope-presenting APCincreases the concentration of cells and therefore the sensitivity ofthe detection. However, an in vitro re-stimulation is not feasible forcertain applications in which there is to be discriminated betweenactivated and non-activated T-cells, like for example in the case of thedetection of auto-aggressive T-cells if multiple sclerosis is suspected.

The di- and tetramer technologies are new and elegant methods for thedetection of epitope-specific CD8⁺ and CD4⁺ T-cells. However,restrictions of these methods concerning a broad application in theT-cell diagnostics reside in the very high expenses for the productionof the di- and tetramers. In addition, the di- and tetramer technologyis presently only available for a limited number of MHC types,especially for MHC-class I proteins which are frequently represented inthe population. Moreover, this technique only allows the detection ofdefined epitope-specific T-cells with known MHC-restriction. Theexamination of T-cell reactivities against multiple epitopes with thismethod is only feasible with a great expense of time and costs.

Therefore, the present invention is based on the problem to provide amethod for the identification of epitopes which are specificallyrecognised by reactive T-cells.

Furthermore, the present invention is based on the problem to providemethods for the detection of epitope-specific T-cells.

The problem is solved by the subject-matter defined in the patentclaims.

The following figures are to illustrate the invention.

FIG. 1 show in a schematic view the arrangement of the promoters and thefunctionally linked nucleic acids in the vector according to theinvention. (P₂) denotes a promoter being constitutively active in APC,(PP) denotes any polypeptide, (P₁) denotes a promoter being inducible inAPC by the epitope-specific contact with a T-cell, (M) denotes a marker,(L) denotes a signal peptide, (LY) denotes an endo/lysosomal targetingsignal, (TD) denotes a heterologous transmembrane domain, RE denotesrestriction sites, ori means the origin of replication and R meansresistance gene.

FIG. 2 exemplarily shows the vector backbone pcDNA3.1(+)-Z according tothe invention for vector backbones concerning the expression ofpolypeptides under the control of a promoter being constitutively activein APC (P₂). This plasmid backbone contains the coding region of theEpstein-Barr virus BZLF-1 protein under the transcriptional control ofthe cytomegalo virus (CMV)-promoter which is constitutively active inAPC.

FIG. 3 exemplarily shows the vector backbone pcDNA3.1(+)-Z/LAMPaccording to the invention for vector backbones which mediate a targetedtransport into the endolysosom of the polypeptides which have beenexpressed by means of the constitutive promoter (P₂). This plasmidbackbone contains the coding region of the Epstein-Barr virus BZLF-1protein which is linked to the coding region of the LAMP-1 signalpeptide in the 5′-region and with the coding region of the transmembraneand cytoplasmatic domain of the human LAMP-1 protein in the 3′-region.The coding region of the chimeric protein is under the transcriptionalcontrol of the cytomegalo virus (CMV)-promoter which is constitutivelyactive in APC.

FIG. 4 exemplarily shows the vector backbone pcDNA3.1(+)-Z/TM accordingto the invention for vector backbones which ensure a membrane anchoringof the polypeptides which have been expressed by means of theconstitutive promoter (P₂). This plasmid backbone contains the codingregion of the Epstein-Barr virus BZLF-1 protein which is linked to thecoding region of the LAMP-1 signal peptide in the 5′-region and with thecoding region of the transmembrane domain of the Epstein-Barr virusgp220/350 coat protein in the 3′-region. The coding region for thechimeric protein is under the transcriptional control of the cytomegalovirus (CMV)-promoter which is constitutively active in APC.

The FIGS. 5A-D exemplarily show four vector backbones according to theinvention comprising a first promoter (P₁) which is specificallyinducible in antigen-presenting cells by the epitope-specific contactwith a T-cell, a nucleic acid being functionally linked to this firstpromoter and encoding a marker gene, a second promoter (P₂) which isconstitutive in antigen-presenting cells, and a nucleic acid beingfunctionally linked to this second promoter.

FIG. 5A shows the vector backbone pcDNA3(+)OX40LAeGFP according to theinvention. This plasmid backbone contains the gene for the enhancedgreen fluorescent protein (eGFP) as a marker under the transcriptionalcontrol of the OX40-ligand promoter which is inducible in APC after anepitope-specific recognition by a T-cell.

FIG. 5B shows the vector pcDNA3(+)OX40LAeGFP-Z according to theinvention. This plasmid contains the gene for eGFP as a marker under thecontrol of the inducible OX40-ligand promoter. In addition, this plasmidcontains the coding region of the Epstein-Barr virus BZLF-1 proteinunder the transcriptional control of the cytomegalo virus (CMV)-promoterwhich is constitutively active in APC.

FIG. 5C shows the vector pcDNA3(+)OX40LAeGFP-MBP according to theinvention. This plasmid contains the gene for eGFP as a marker under thecontrol of the inducible OX40-ligand promoter. In addition, this plasmidcontains the coding region of the human myelin basic protein (MBP) underthe transcriptional control of the cytomegalo virus (CMV)-promoter whichis constitutively active in APC.

FIG. 5D shows the vector pcDNA3.1(+)Z/LAMP/OX40L according to thepresent invention. This plasmid backbone contains the coding region ofthe Epstein-Barr virus BZLF-1 protein which is linked to the codingregion of the LAMP-1signal peptide in the 5′-region and with the codingregion of the transmembrane domain of the Epstein-Barr virus gp220/350coat protein in the 3′-region. The coding region of the chimeric proteinis under the transcriptional control of the cytomegalo virus(CMV)-promoter which is constitutively active in APC. In addition, thisplasmid contains the gene for eGFP as a marker under the control of theinducible OX40-ligand promoter.

The FIGS. 6A-B show the efficiency of the plasmid transfer into APC bymeans of the Nucleovector™ technology. Hereby either populations of CD3⁺depleted PBMCs (FIG. 6A) or of purified B-cells (FIG. 6B) were preparedby means of the Miltenyi cell separation technology and the compositionof the obtained cell populations was determined by means of the FACStechnology using suitable antibodies (FIGS. 6A,B; left panel each).3×10⁵ purified CD3⁺ cell-depleted PBMC or isolated B-cells each weresubsequently transfected with 5 μg of the vector pcDNA3.1(+)dNeo-eGFPaccording to the present invention (FIGS. 6A,B; right panel each) and asa control with the pcDNA3.1(+) vector (FIGS. 6A,B; middle panel each)using the Nucleovector™ technology. The efficiency of the transfer ofthe nucleic acids in the denoted cell populations was determined by themeasurement of the number of fluorescent cells (detection of aGFP-reporter) after 17 hours after transfection using a FACS device.

FIG. 7 shows the nucleic acid sequence of the human OX40-ligand promoterwhich was amplified by PCR (SEQ ID NO: 1).

The FIGS. 8A-D show the nucleic acid sequences of the human 4-1BB-ligand promoter (4-1 BBL) which was amplified by PCR and of threevariants of the human 4-1 BB-ligand promoter with progressivetruncations in the 5′-region. FIG. 8A: long variant (V1) (SEQ ID NO: 2);FIG. 8B: C-terminally truncated variant (V2) (SEQ ID NO: 3); FIG. 8C:C-terminally further truncated variant (V3) (SEQ ID NO: 4); FIG. 8D:C-terminally even further truncated variant (V4) (SEQ ID NO: 5). TheBglII restriction site which is present in the sequences is marked. Thisrestriction side was mutagenised by a G to A exchange. The herein usedterm “genomic” denotes the collectivity of fragments of the geneticmaterial of an organism.

The herein used term “polynucleotide” denotes the polymeric form ofnucleotides of any length, preferentially desoxyribonucleotides (DNA) orribonucleotides (RNA). This term denotes only the primary structure ofthe molecule. The term comprises double- and single stranded DNA and RNAas well as antisense-polynucleotides.

The herein used term “control sequences” denotes polynucleotidesequences which are necessary for the expression of codingpolynucleotide sequences, they are linked to. Control sequences arepresent in the genome of organisms and regulate the transcription ofgenes, i.e. the synthesis of mRNA. Control sequences are present also onmRNA-polynucleotides and regulate the translation, i.e. the synthesis ofpolypeptides. The features of such control sequences vary in thedependency on the host organism; in the case of prokaryotes such controlsequences contain usually a promoter, a ribosomal entry site and atranscription termination sequence; in eukaryotes such control sequencesusually contain promoters and transcription termination sequences. Inaddition, the term “control sequence” comprises all polynucleotideswhose presence is necessary for the constitutive or inducible expressionof coding sequences and moreover includes additional components whosepresence is beneficial for the expression of a polypeptide, like forexample leader sequences and the sequences of a fusion partner.

The herein used term “polypeptide” denotes a polymer of amino acids ofany length. The term polypeptide comprises also the terms (target-)epitope, peptide, oligopeptide, protein, polyprotein, and aggregates ofpolypeptides. Likewise, this term includes polypeptides which showpost-translational modifications like for example glycosylations,acetylations, phosphorylations and similar modifications. Furthermore,this term comprises for example polypeptides which show one or moreanalogues of amino acids (e.g. unnatural amino acids), polypeptides withsubstituted linkages as well as other modifications which are state ofthe art, independent of the fact whether they occur naturally or if theyare of non-natural origin.

The herein used term “epitope” denotes the region of a polypeptideexhibiting antigenic features and serving for example as a recognitionsite of T-cells or immunoglobulins. In terms of this invention epitopesare for example such regions of polypeptides which are recognised byimmune cells like for example CD4⁺ T-helper cells, CD8⁺ cytotoxicT-cells, CD161⁺ 0 NKT cells or CD4⁺CD25⁺ regulatory T-cells. An epitopecan comprise 3 or more amino acids. Usually an epitope consists of atleast 5 to 7 amino acids or, more often, of at least 8-11 amino acids,or of more than 11 amino acids, or of more than 20 amino acids, lessfrequently even of more than 30 amino acids. The term “epitope”comprises both linear and a steric conformation being unique for theepitope. The steric conformation results from the sequence of the aminoacids in the region of the epitope.

The herein used term “micro-organism” denotes viruses as well asprokaryotic and eukaryotic microbes such as archae-bacteria, bacteria,protozoa and fungi; the latter group comprises for example yeast andfilamentous fungi.

The herein used term “vector” or “gene-transfer vector” denotesnaturally occurring or artificially generated organisms and constructsfor the uptake, propagation, expression or transfer of nucleic acids incells. Vectors are for example viruses such as lenti viruses, retroviruses, adeno viruses, adeno-associated viruses, pox viruses, alphaviruses, baculo viruses, rabies viruses or herpes viruses. Vectors arefor example also bacteria such as listeriae, shigellae or salmonellae.But vectors are for example also naked DNA such as bacterial plasmidsand MIDGES, virus derived plasmids, phagemids, cosmids, bacteriophagesor artificially generated nucleic acids such as artificial chromosomes.Vectors are able to propagate autonomously inside a cell. Furthermore,the vector can contain one or more additional polynucleotides in such away that these can be replicated and/or expressed. Moreover, vectors cancontain one or more selection marker(s).

T-cells in terms of the invention are lymphocytes with regulatory orcytolytic features like for example CD4⁺ T-helper cells, CD161⁺ NKTcells, CD8⁺ cytotoxic T-cells and CD4⁺CD25⁺ regulatory T-cells.

The used term “antigen presenting cell” (APC) comprises cells which areable to take up polypeptides, to process them and to present fragmentsof these polypeptides (epitopes) to the immune system in connection withMHC I and MHC II proteins. In particular, the term “antigen presentingcell” comprises dendritic cells (Langerhans cells), monocytes,macrophages, B-cells but also vascular endothelial cells and differentepithelial, mesenchymal cells as well as microglia cells of the brain.

The term “linked” denotes an attachment by means of covalent bonds orstrong, non-covalent interactions (e.g. hydrophobic interactions,hydrogen bonds, etc.). Covalent bonds can be for example ester, ether,phosphor-ester, amides, peptides, imides, carbon-sulphur bonds,carbon-phosphate bonds or similar bonds.

One aspect of the present invention relates to vectors comprising afirst promoter (P₁) which is specifically inducible in APC by theepitope-specific contact with a T-cell, a nucleic acid encoding a markergene and being functionally linked to said first promoter, a secondpromoter (P₂) which is constitutive in APC, and a nucleic acid which isfunctionally linked to said second promoter. Preferably, the vectorcontains preferably a bacterial origin of replication (ori) and aresistance gene. Furthermore, the vector according to the invention maycontain suitable recognition sequences for restriction endonucleasesflanking the constitutive second promoter (P₂), the nucleic acidsequence which is functionally linked to this second promoter as well asthe bacterial origin of replication and the coding sequence for thebacterial resistance. Furthermore, the vector according to the inventioncan contain a third promoter which is functionally linked to yet anothermarker gene.

The polynucleotides encoding a marker are under the control of apromoter (P₁) which is inducible in APC due to an epitope-specificrecognition by a T-cell. In terms of the invention suitable markers arefor example, but not limited to, easily detectable polypeptides orfragments of polypeptides as well as arbitrarily modified derivativesthereof, which can be detected by simple enzymatic reactions orimmunological techniques or because of their fluorescence. Examples forauto-fluorescent markers are the Vitality™ human recombinant (hr)GFP(Stratagene, Amsterdam, The Netherlands), the “green-fluorescentprotein” (GFP) (BD Clontech, Heidelberg, Germany), FACS-optimisedvariants of the GFP, the “blue-fluorescent protein” (BFP), the “DsRedfluorescent protein” (BD Clontech), the “red-fluorescent protein” (RFP),the “yellow-fluorescent protein” (YFP), the “cyan fluorescent protein”(CFP) (BD Clontech) or derivates of these proteins, which show anincreased fluorescence, like the “enhanced green-fluorescent protein”eGFP (BD Clontech), the “enhanced blue-fluorescent protein” eBFP, the“enhanced red-fluorescent protein” eRFP, the “enhanced cyan-fluorescentprotein” eCFP (BD Clontech) or the “enhanced yellow-fluorescent protein”eYFP (BD Clontech). Examples for markers with enzymatic activity are forexample the luciferase (LUC) (BD Clontech), the alkaline phosphatase(AP) (BD Clontech), the secretory alkaline phosphatase (SEAP) (BDClontech), the chloramphenicol acetyltransferase (CAT) (Promega,Mannheim, Germany), the photinus-luciferase (BD PharMingen), theβ-glucuronidase (GUS) (Research Diagnostics, New York, USA), therenilla-luciferase (Promega) and the β-galactosidase (β-Gal) (BDClontech). Examples for markers which can be easily detected by means ofimmunological methods are in addition any intra-cellular andmembrane-associated polypeptides which do not occur naturally in the APCbeing modified by the vectors according the invention and which aredetectable by means of common immunological or biochemical methods forexample by means of polypeptide-specific antibodies. Therefore, themembrane-associated murine proteins CD4, CD5, CD8a, CD11b, CD11c, CD19,CD43, CD45, CD62L, CD90, are for example suited as markers, but notlimited to, but also the proteins CD4, CD8a, CD45, CD45RA and CD134(OX40) of the rat as far as they do not display an interferingcross-reactivity with the respective human surface proteins.Analogously, the human surface proteins like for example CD2, CD3, CD4,CD8, CD11b, CD14, CD15, CD16, CD19, CD22, CD27, CD30, CD45RO, CD45RA,CD56, CD69, CD138 are suited as markers for the detection of targetepitopes of reactive T-cells by means of the method according to theinvention in the case of non-human vertebrates. For the detection of allthese proteins by the FACS-technology fluorescence-linked primary andsecondary antibodies are suited (for example R-phycoerythrin (R-PE),peridin-chlorophyll c (PerCP), fluorescein (FITC), Texas Red (TX),allophycocyanin (APC), Tandem PE-TX, Tandem PE-Cy5, PE-Cy7, TandemAPC-Cy7). These are either commercially available (for example from thecompanies Becton Dickinson, Dako, Coulter) or they can be generated withcommercially available kits following the manufacturer's protocol.Moreover, for example microbial, in particular viral coat proteins, likefor example, but not limited to, the coat proteins of the humanimmune-deficiency virus (HIV), of the simian immune-deficiency virus(SIV), of the human T-cell leukaemia viruses (HTLV) and of the vesicularstomatitis virus (VSV) are suited as markers. But also coat proteins andstructural proteins of any virus and bacterium are suited as markers.However, the test persons/patients to be analysed must not exhibit anynatural antibodies against these marker proteins in their serum.

In addition, polypeptides are suited as markers which exhibit celltransforming features. Examples for such polypeptides are viral proteinslike the adenoviral proteins E1A and E1B, the E6 and E7 proteins of thehuman papilloma virus, the great T-antigen of the SV40 virus, the LMP-1protein of the Epstein-Barr virus (EBV) as well as the X antigen of thehepatitis B virus. In addition, other non-viral polypeptides withtransforming features can be used, too.

In particular polypeptides are suited as markers whose expression ismeasurably increased as a result of an epitope-specific recognition ofan APC by a T-cell in the APC. Examples for these polypeptides are theOX40-ligand (OX40L) and the 4-1 BB-ligand (4-1 BBL) (Ohshima et al.,1997, J. Immunol. 159, 3838-3848; den Haan and Bevan; 2000, PNAS 97,12950-12952). In addition, the co-stimulatory proteins B7.1 (CD80), B7.2(CD86) and the Fas ligand (FasL) are suited. In addition, in terms ofthe present invention any polypeptide is suited whose expression ismeasurably increased or reduced as a result of a specific recognition ofan epitope being presented together with MHC proteins on the surface APCin the same APC.

Furthermore, polypeptides are also suited as markers which weregenerated by any combination of different naturally occurringpolypeptides as well as polypeptide sequences which do not occurnaturally. These polypeptides are suited as markers if they aredetectable by immunologic, (bio)chemical or physical methods in thecytoplasm or on the surface of APC which have been modified by thetransfer systems according to the invention.

The expression of the marker is under the control of a promoter (firstpromoter, P₁) which is inducible by the epitope-specific contact with aT-cell. In terms of the invention, hereby any promoter is suited whichis turned on or shows a significant increase of its activity as a resultof a specific interaction of the T-cell receptor (TCR) of a specificT-cell with a peptide which is presented in connection with MHC proteinsin the APC on the surface of the APC which have been modified by thevector according to the invention. Examples of such promoters arepromoters for the OX40-ligand (OX40L) and of the 4-1 BB ligand (4-1 BBL)(den Haan and Bevan, 2000). In addition, the promoters for theco-stimulatory proteins B7.1 (CD80), B7.2 (CD86) and the promoter forthe Fas ligand (FasL) are suited. In addition, any promoter is suited interms of the invention which is turned on or shows a measurable increasein its activity as a result of the specific recognition of an epitopewhich is presented together with MHC proteins on the surface of APC by aspecific T-cell in the same APC.

Furthermore, suitable promoters in terms of the invention are such oneswhich are turned off or are measurably reduced in their activity as aresult of a specific interaction of the T-cell receptor (TCR) of aspecific T-cell with a peptide being presented in the APC on the surfaceof the APC which have been modified by the vectors according to theinvention in connection with MHC proteins. Hereby the reduction of theexpression of a polypeptide being naturally produced by APC but also thereduction of the expression of a marker in the APC as a result of anepitope-specific contact with a T-cell can serve for a selectioncriterion.

The vectors according to the invention encode a marker or a combinationof two or more different markers which are under the control of the sameor different inducible first promoters as described above. If two ormore markers are present on the vectors according to the invention, onlythe first marker gene has to be under the transcriptional control of apromoter which is inducible in APC. The expression of further markersunder the control of a constitutive promoter is for example suited forchecking the transfection/transformation-efficiency of the APC whichwere treated with the vectors and transfer systems, respectively,according to the invention.

Furthermore, the vector according to the invention contains a secondpromoter (P₂) which brings about a constitutive expression of thenucleic acid, being functionally linked to it, in the APC. Genes ofviruses that infect mammals are often expressed very efficiently inmammalian cells and show a broad host range. Therefore, the respectiveviral control sequences are particularly suited as promoter P₂ for theexpression of gene sequences in mammalian cells. Some representatives ofsuited viral control sequences are for example the early SV40 promoter,the cytomegalo virus (CMV) promoter, the respiratory syncytial virus(RSV) promoter, the mouse mammary tumour virus (MMTV) LTR promoter, thehuman immune-deficiency virus type 1 (HIV-1) LTR promoter, the adenovirus major late promoter (Ad MLP) and the herpes simplex virus (HSV)promoter, and the promoter sequences derived thereof. Furthermore, alsopromoters of non-viral genes, like e.g. the murine3-phosphoglycerate-kinase (PGK) promoter, the human PGK-1 promoter, thehuman ubiquitin C promoter, the human EF-1α promoter, the human β-caseinpromoter, the murine metallo-thioneine promoter, the human actin 5cpromoter or the human ICI promoter are suited for the efficientexpression of polynucleotides in mammals. If the control sequences (1)for the constitutive expression of the polypeptide and the (2) inducibleexpression of the marker are present on one vector, only such controlsequences are suited for the constitutive expression of the polypeptidewhich do not interfere with the functionality according to the inventionof the control sequences for the inducible expression of the marker.

Both functional regions (1) for the constitutive expression of thepolypeptide and the (2) inducible expression of the marker are usuallypresent on one vector. However, these both functional regions can bepresent also on separate vectors which are then co-introduced in oneAPC.

The nucleic acid which is functionally linked to the second constitutivepromoter, encodes polypeptides which represent or contain known orputative targets of reactive T-cells. Thereby, the nucleic acid canexhibit a naturally occurring sequence or an arbitrary sequence.However, the nucleic acid sequence can also be derived for example fromany genomic or cDNA library.

If the vectors according to the invention are supposed to be used in thesearch for epitopes, any nucleic acids encoding polypeptides withunknown, potential target epitopes of reactive T-cells, like for examplepolynucleotides from a cDNA library or a genomic library, can be clonedunder the control of the constitutive promoter (P₂). The cDNA librarycan be for example a species-specific, a pathogen-specific, atissue-specific, a development-specific or a subtractive library. Alimitation concerning the size of the cloned polynucleotides does notexist. The cloned polynucleotides can exert any length, e.g. they cancomprise less than 20 nucleotides or, occurring more frequently, 20 to100 nucleotides, but also 100 to 500, 500 to 1,500 nucleotides, but alsoup to 5,000 nucleotides, more rarely up to 10,000 nucleotides, but alsomore than 10,000 nucleotides.

If the vectors according to the invention are to be used for thedetection of epitope-specific T-cells, nucleic acids encodingpolypeptides with known target epitopes of reactive T-cells can becloned under the control of the constitutive promoter (P₂). The clonedpolynucleotides can exert any length, e.g. they can comprise less than20 nucleotides, or, occurring more frequently, 20 to 30 nucleotides, butalso 30 to 100 nucleotides, 100 to 500 nucleotides, but also up to 1,000nucleotides, more rarely up to 10,000 nucleotides.

The used arbitrary or known nucleic acids can be derived from humans ormammals, but also from any animals, parasites or micro-organisms, e.g.bacteria or viruses. But they can be derived from plants or algae, too.Moreover, they can be derived from prion proteins.

Examples for viruses whose polypeptides or fragments are encoded by thenucleic acid sequences being comprised by the vector according to theinvention are listed in the following. Particularly significant viruseswith partly (human) pathogenic features are for example, but not limitedto, polio viruses, coxsachie viruses, echo viruses, entero viruses,rhino viruses, orthomyxo viruses (especially type A, B, C influenzaviruses), paramyxo viruses (especially para-influenza viruses, mumpsviruses, measles viruses, respiratory syncytial viruses (RS-virus),corona viruses, flavi (especially yellow fever, dengue, JapanB-encephalitis, tick-born encephalitis (FSME) viruses), the hepatitis Cvirus (HCV)), toga (especially alpha- and rubidi viruses) and bunya(especially the bunya, hanta, nairo, phlebo and tospo virus genera)viruses, rubella viruses, rabies viruses, arena viruses (especially thelymphocytic chorio-meningitis virus (LCMV) and the Lassa fever virus),gastroenteritis viruses (especially rota viruses, adeno viruses, caliciviruses, astro viruses, corona viruses), retro viruses (especially typeA, B, C and D retro viruses, lenti viruses (especially the humanimmune-deficiency viruses type-1 (HIV-1) and -2 (HIV-2)), the simianimmune-deficiency virus (SIV), the feline immune-deficiency virus (FIV),and the bovine immune-deficiency virus (BIV)), spuma viruses, the humanT-cell leukaemia viruses type-1 (HTLV-1) and -2 (HTLV-2)), parvo viruses(especially parvo virus B 19 and adeno associated viruses (AAV)), papovaviruses (especially papilloma viruses, the virus of the progressivemultifocal leukoencephalopathy (PML), BK-virus), adeno viruses, herpesviruses (especially the herpes simplex virus type-1 (HSV-1) and -2(HSV-2), the varicella-zoster virus (VZV), the cytomegalo virus (CMV)and the Epstein-Barr virus (EBV), the human herpes viruses 6, 7 and 8(HHV 6, 7 and 8), hepatitis viruses (especially the hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis Cvirus (HCV), hepatitis E virus (HEV) and hepatitis G virus (HGV) as wellas the transfusion-transmitted virus (TTV)) and pox viruses (especiallyorthopox viruses (like the human pox-virus, vaccinia viruses, cow poxviruses and para-pox viruses)). Furthermore, the polypeptide can bederived from viral pathogens which elicit rare sub-acute or chronicdiseases (especially the Marburg and ebola viruses as well as the bomaviruses).

For example, the polypeptides can be derived from important viruseswhich are pathogenic for animals. Significant representatives of viruseswhich are pathogenic for animals are for example, but not limited to,the equine morbilli virus (EMP), picoma viruses (especially enteroviruses, aphto viruses (with the elicitor of the foot and mouth disease(FMD)), the vesicular stomatitis virus, paramyxo viruses (especiallymorbilli viruses, avian paramyxo viruses), pox viruses (especiallycapripox viruses), bunya viruses, reoviruses (especially orbi viruses),flavi viruses (especially pesti viruses), orthomyxo viruses (especiallythe influenza A virus), herpes viruses (especially alpha herpesviruses), rabies viruses, retro viruses (especially lenti viruses andC-type retro viruses), toga viruses, rhabdo viruses, bima viruses,corona viruses and calici viruses.

An all-embracing listing of viruses which are described at present wasfor example assembled by the international committee on taxonomy ofviruses (ICTV) and is accessible via the internet(http://www.ncbi.nlm.nih.gov/ICTV/).

Examples for bacteria whose polypeptides or fragments are encoded by thenucleic acid sequences which are comprised by the vector according tothe invention are listed in the following. In principal the polypeptidescan be derived of any bacterium. However, they are preferentiallyderived from intracellular bacteria. Significant intracellular bacteriaare for example, but not limited to, listeriae (especially L.monocytogenes), salmonellae (especially S. typhimurium) andmycobacteriae (especially M. tuberculosis).

Furthermore, the polypeptides can be derived from human pathogenicbacteria. Significant representatives of human pathogenic bacteria arefor example, but not limited to, staphylococcae, streptococcae,enterococcae, neisseriae, enterobacteriae (especially Escherichia coli(E. coli), inclusively E. coli strains which are pathogenic for babies(EPEC), enteroaggregative E. coli strains (EAggEC), clebsiellae,enterobacter, serratia, proteus, citrobacter and typhoid salmonellae),enteritis salmonellae, shigellae, yersiniae), vibrionae (especiallyVibrio cholerae und Vibrio El Tor), pseudomonades, burkholderia,stenotrophoma, acinetobacter, campylobacter, helicobacter (especiallyHelicobacter pylori), haemophilus, bordetellae, legionellae, listeriae,brucellae, francisellae, erysipelothrix, korynebacteriae, bacillus,clostridiae, bacteroides, prevotellae, porphyromonae, fusobacteriae,anaerobiospirillae, anaerorhabdus, anaerovibrio, butyrivibrio,centripedia, desulfomonas, dichelobacter, fibrobacter, leprotricha,megamonas, mitsuocella, ricenella, sebaldella, selenomonas,succinovibrio, succinimonas, tisserella, mycobacteria (especially M.tuberculosis, atypic mycobacteria (MOTT) and M. leprae), nocardia,treponema (especially T. pallidum and T. carateum), borreliae(especially B. burgdorferi and B. recurrentis), leptospirae,ricksettsiae, coxiellae, ehrlichiae, bartonellae, mycoplasma (especiallyM. pneumoniae and M. hominis), ureaplasma, actinomycetes, chlamydiae. Inaddition, the polypeptides can be derived from further medicallysignificant bacteria like for example tropheryma, pasteurella,branhamella, streptobacillus, spirillum and gardnerella.

Furthermore, the polypeptides can be derived from bacteria which arepathogenic for animals. Significant representatives of bacteria whichare pathogenic for animals are for example, but not limited to,mycoplasma, bacillus (especially Bacillus anthracis), brucellae,mycobacteriae (especially M. tuberculosis and M. bovis), campylobacter,tritrichomona, leptospirae, rickettsiae, salmonellae, clostridiae,actinobacillae, clamydiae, echinococcae, listeriae, yersiniae,corynebacteriae und francisella.

Examples for fungi whose polypeptides or fragments are encoded by thenucleic acid sequences which are comprised by the vector according tothe invention are listed in the following. Particularly significant(human) pathogenic fungi are for example, but not limited to, blastomyyeasts (especially candida, cryptococcus, malassetia), hyphal yeasts(especially aspergillus, trichphyton, microsporum, and epidermophyton),dimorphic fungi (especially histoplasma, blastomyces, coccidioides,paracoccidioides, sporothrix) and pneumocystis.

Examples for parasites whose polypeptides or fragments are encoded bythe nucleic acid sequences which are comprised by the vector accordingto the invention are listed in the following. Significantrepresentatives of human pathogenic parasites are in particular alsoprotozoa like tryphanosoma, leishmania, trichomona, giardia, amoebae,plasmodia, toxoplasma, cryptosporidia, microsporidia. Significantrepresentatives of human pathogenic parasites are in particular alsotrematodes like for example shistosoma as well as cestodes like forexample tape worms and echinococcae as well as nematodes like forexample trichuris, trichinella, strongyloides, ancyclostoma, necator,enterobius, ascaris and filaria. Significant representatives ofparasites being pathogenic for animals are for example, but not limitedto, protozoa (especially protomonades, diplomonades, polymastigida,amoebae, toxoplasms, coccidia), mirospores, helminthes, trematodes,cestodes and nematodes.

The polynucleotides encoding the polypeptides of humans, primates, othermammals, any other animals, parasites, micro-organisms, plants, algae oralso polynucleotides encoding prion proteins can, in addition, be linkedto each other in any manner.

But they can be also linked to any nucleic acids which encode functionalpolypeptides. Polypeptides with functional features are for examplesignal peptides for the polypeptide targeting into the endoplasmaticreticulum (ER), for example the signal peptides of mellitin,erytropoetin, the human interleukin-3, the human interleukin-8, thehuman LAMP-1 and -2 protein or the tPA signal sequence. But suchpolypeptides are also endo- and lysosomal signal sequences orpolypeptides having endo- and lysosomal signal sequences like forexample the cytoplasmatic region of the Ii chain or regions of the“invariant chain”, or of LAMP-1, LAMP-2, LIMP-1 (CD63), LAP or MHC-classII proteins which contain endo/lysosomal signal sequences.

Such polypeptides are, in addition, polypeptides which representtransmembrane domains for example, but not limited to, the transmembranedomain of the Epstein-Barr virus gp220/350 coat protein, of the HIV gp41transmembrane protein, but also any other transmembrane domains of othernaturally occurring transmembrane proteins, but also syntheticallygenerated transmembrane domains. The vectors according to the inventioncontain usually a bacterial origin of replication and a gene whichbrings about a resistance against for example ampillicin, kanamycin,tetracyclin or zeocin. The coding regions of the bacterial origin ofreplication and the antibiotic resistance are preferably located inproximity to the expression unit for the constitutive expression ofpolypeptides (P₂) with known or putative T-cell epitopes. The nucleicacids which are necessary for the constitutive expression of apolypeptide, the origin of replication and the resistance gene, can beflanked, on the vector according to the invention, on both sides by oneor more recognition sequence(s) for restriction sites whichpreferentially is/are not present within the bordered nucleic acidsequence including the sequence for the origin of replication, theresistance gene and the expressing unit for the constitutive expressionof the polypeptide with the known or putative T-cell epitopes. This partof the sequence on the vector can also be bordered by two different,however compatible, restriction sites for restriction endonucleasesunless both restriction sites are present within the nucleic acidsequence comprising the origin of replication, the resistance gene andthe expression unit for the constitutive expression of the polypeptide.The presence of these restriction sites for restriction enzymes is notrequired if episomaly available plasmids are used as a transfer systemfor the vectors according to the invention.

Furthermore, the vector according to the invention can contain thefollowing control sequences which are known to a person skilled in theart and which belong to the state of the art: transcription-terminationand polyadenylation sequences, enhancer, introns with functional donorand acceptor sites for splicing as well as leader sequences, a TATA-box,a GC-box, a CAAT-box and other promoter elements which are usuallylocalised upstream of the TATA-box as well as an optimal but alsosub-optimal Kozak-sequence.

Expression cassettes are often contained within a replicon, like e.g. inextra-chromosomal elements (e.g. plasmids), being able to persist stablyin a host like e.g. in a mammalian cell. Mammalian replication systemscontain cassettes being derived from animal viruses, for example papovaviruses, polyoma viruses, bovine papilloma viruses or from theEpstein-Barr virus and which require trans-active factors for thereplication.

Moreover, the expression efficiency of the desired foreign gene can beincreased by the choice and use of suited, host specific codons. Thisobservation is based on the finding that both prokaryotic and eukaryoticgenes do not exhibit a static usage of synonymous codons.

As vector backbones for the generation of plasmids according to theinvention for example, but not limited to, pcDNA expression vectors aresuited which are based on the prototype plasmid pBR322 (Bolivar et al.in: DNA Insertion Elements, Plasmids and S. Episomes. Bukhari, A.,Shapiro J. A., and Adhaya S. L. (eds.) Cold Spring Harbor Laboratory,USA, pp. 686-687, (1977)) which allow an efficient gene expression inmammalian cells.

Furthermore, a multiplicity of different non-viral and viral genetransfer vectors is suited to introduce nucleic acids into mammaliancells. Some of the most significant vector systems for the geneexpression in mammalian cells are listed in the review by Makrides(Makrides, Protein Expr. Purif. (1999), 17(7), 183-202). Many of thedescribed vectors are particularly suited for the transduction ofnucleic acids in APC and commercially available.

For example, plasmid DNA, in particular also the vectors according tothe invention, can be used directly for the transduction of cells. Inparticular, a second generation of linear DNA plasmids, the so-calledMIDGE-transfection vectors (Mologen AG, Berlin, Germany), is suited forthe efficient transfer of the nucleic acids described in this patentspecification. Different forms of application for the transduction ofmammalian cells (for example, but not limited to, the lipofection,electroporation or calcium phosphate precipitation) as well as for theenhancement of the efficiency of the uptake of vectors are published andstate of the art. Particularly suited for the transfer of nucleic acidsin APC are for example the Femtosecond Laser Technology protocol(Tirlapur and Konig, (2001), Nature 418, 290), the Nucleovector™technology (Amaxa, Germany) or the Fugene 6 Transfection reagent (Roche,Germany).

Moreover, the efficiency of the transfer of nucleic acids can bedrastically enhanced by the use of miniature liposomes with a diameterof about 25 nm (Copernicus Therapeutics, USA).

The efficiency of the gene transfer via retro-viral transfer systems cane.g. be enhanced by adjusting the target cells in the S phase. Inaddition, the methods for optimising the efficiency of the transductionof mammalian cells via viral transfer systems comprise the variation ofthe “multiplicity of infection” (M.O.I.), the depletion of ions likee.g. phosphate ions, the addition of polycationic substances, forexample of protamin sulphate, the variation of the period of contact,temperature, the pH value, a co-centrifugation of the cells and thevirus and vector stocks, respectively, or the incubation of the cellswith the transfer systems in a small volume of medium.

The transfer systems according to the invention which have beengenerated in such a way are both suited for the search for epitopes andfor the detection of epitope-specific T-cells.

Therefore, the invention relates to the provision of gene transfervectors for the modification of APC by means of these vectors. Thevectors according to the invention transfer a combination of twocharacteristics onto the APC:

-   -   (1) The APC obtain the ability to constitutively and        intracellularly produce single known target structures, e.g. in        the case of an application in diagnostics, or still unknown        potential target structures, e.g. in the case of the search for        epitopes, and to present fragments thereof in connection with        MHC-proteins of the classes I and/or II on their surface.    -   (2) In the case of the recognition of the target structure        (epitope) presented on the surface of the APC by a specific        T-cell, the expression of the marker is induced or inhibited via        the signal in the APC which was elicited by the T-cell. The        changed expression of the marker, and by this means the        activated APC, can be reliably and rapidly detected in a simple        and quantitative manner, for example by means of commercially        available devices and methods (for example FACS-,        immuno-fluorescence-, ELISA-, ELISPOT-technology) and be        isolated (for example by FACSsorting, magnetic cell-sorting).

The APC which have been modified by means of the vectors according tothe invention are suited for a quick detection and characterisation ofup to now unknown target structures of T-cells and thus represent asuitable tool for the construction of disease- or patient-specificdatabases. In addition, the APC which have been modified by the vectorsaccording to the invention can be used for the detection of any T-cellpopulations which have known target structures. The methods according tothe invention apply to all vertebrates which have T-cells, in particularto humans, primates and rodents. The knowledge of up to now unknowntarget regions of activated T-cells, in addition, opens up newperspectives concerning the development of novel concepts indiagnostics, therapy and vaccination for the prophylaxis and treatmentof pathogen-induced diseases, auto-immune diseases, transplantrejections and chronic inflammatory diseases. Moreover, the knowledge ofT-cell epitopes in non-human vertebrates opens up the establishment ofnew animal model systems for research purposes.

A further aspect of the present invention relates to antigen presentingcells (APC) being transduced with the vectors according to theinvention. The generation of APC according to the invention takes placeby the treatment of APC with the gene transfer vectors according to theinvention. The treatment of the APC with the gene transfer vectorsaccording to the invention can take place by the incubation ofheparinised whole blood or defined peripheral blood-mononucleated cells(PBMC) which were purified from the whole blood, as well as purifiedpopulations of defined APC (for example B-cells, dendritic cells,monocytes, macrophages) with viral and bacterial gene transfer vectorsaccording to the invention which exhibit a specific tropism for all APCor for defined sub-populations of APC. Alternatively, in particularpurified populations of PBMC and purified populations of defined APC(for example B-cells, dendritic cells, monocytes, macrophages) aresuited for the treatment with the non-viral gene transfer vectorsaccording to the invention, for example with plasmids, MIDGE vectors andreplicons. These non-viral vectors are preferentially introduced intothe target cells by means of physical/chemical methods (for exampleFemtosecond Laser technology, Nucleovector™—technology, transfection bymeans of the Fugene 6 reagent (Roche, Germay), but also by lipofection,or electroporation or calcium phosphate precipitation). Methods for thepurification of PMBC and of defined populations of APC from heparinisedwhole blood as well as the nucleic acid transfer into APC by means ofviral, bacterial as well as non-viral/non-bacterial vectors are state ofthe art.

The success of the transfer of the nucleic acids according to theinvention can be verified by means of a detection of the expression ofthe polypeptide being under the control of the constitutive promoter(promoter P₂) or of the expression of the marker being under the controlof the third promoter by conventional molecular biological andimmunological methods, for example by the immuno-blot, theimmuno-fluorescence or FACS technology with suitable antibodies beingused. Such molecular biological and immunological methods for thespecific detection of polypeptides are multiply published and state ofthe art.

For the generation of APC according to the invention for the search forepitopes, vectors according to the invention are used which exhibitinter alia any polynucleotides being under the control of theconstitutively active promoter (P₂) which encode polypeptides with thepotential target epitopes of reactive T-cells, for examplepolynucleotides of a (subtractive) gene library.

For the generation of APC according to the invention for the detectionof epitope-specific T-cells, transfer systems according to the inventionare used which exhibit inter alia defined polynucleotides being underthe control of the constitutively active promoter (P₂) which encode oneor more polypeptides with known target epitopes of reactive T-cells.

Furthermore, one aspect of the invention relates to APC for the searchfor epitopes or for the detection of epitope-specific T-cells which havebeen transduced with a vector corresponding to the gene transfer vectorsaccording to the invention, with the limitation that said vector doesnot exhibit the expression unit for the marker comprising the promoter(P₁) which is inducible in APC and comprising the functionally linkednucleic acid encoding a marker gene.

A further aspect of the present invention relates to a method for thedetection of epitope-specific T-cells and for the detection of targetepitopes of reactive T-cells comprising the following steps:

-   -   a) Isolation of APC-containing and/or T-cell-containing body        fluid, preferably blood or liquor,    -   b) contacting and transduction of APC-containing body fluid with        gene transfer vectors according to the invention,    -   c) incubation of the body fluid containing the transduced APC or        of isolated transduced APC with the body fluid containing        T-cells or the isolated T-cells, preferably for 0.5 to 36 hours        or longer, particularly preferred for 0.5 to 2, 2 to 6, 6 to 12,        12 to 36 hours or 36 to 168 hours,    -   d) detection of marker expressing APC, and    -   e) optionally, the isolation and characterisation of the nucleic        acid which is functionally linked to the second promoter and        which encodes the target epitopes of reactive T-cells.

Furthermore, the invention relates to a further method for the detectionof epitope-specific T-cells and for the detection of target epitopes ofreactive T-cells comprising the following steps:

-   -   a) Isolation of APC-containing and/or T-cell-containing body        fluid, preferably blood,    -   b) contacting and transduction of APC-containing body fluid with        a nucleic acid inter alia comprising a promoter (P₂) being        constitutive in APC and comprising a nucleic acid being        functionally linked to said promoter,    -   c) incubation of the body fluid containing the transduced APC or        of isolated transduced APC with the body fluid containing the        T-cells or the isolated T-cells, preferably for 0.5 to 36 hours        or longer, particularly preferred for 0.5 to 2, 2 to 6, 6 to 12,        12 to 36 hours or 36 to 168 hours,    -   d) detection of marker expressing APC, and    -   e) optionally, isolation and characterisation of the nucleic        acid which is functionally linked to the second promoter and        encodes the target epitopes of reactive T-cells.

The vectors which are used in step b) of the further method correspondto the gene transfer vectors according to the invention, but have thelimitation that said vectors do not contain the expression unit for themarker comprising the promoter (P₁) which is inducible in APC and thefunctionally linked nucleic acid encoding a marker gene. Besides, theused gene transfer vectors correspond in all functional regions to thegene transfer vectors according to the invention. In fact, in thismethod, instead of the expression units for the marker (P₁) encoded bythe gene transfer vector, available genomic promoters are used which aremeasurably changed in their activity as a result of an epitope-specificrecognition of the APC according to the invention by a T-cell as well asnucleic acids which are functionally linked in a natural way and whichare used as functional units for the marker expression, i.e. inductionor inhibition. The further method also comprises controllable genomicpromoters which are functionally linked to polynucleotides that encodeany user-defined marker.

Preferably, the APC-containing body fluid in step b) of the methods isblood, liquor, the purified PBMC population, or a separated APCpopulation. Preferably, the isolated T-cells are CD4⁺ T-cells, CD8⁺T-cells, CD4⁺ CD25⁺ regulatory T-cells, CD161⁺ NKT cells, or any mixtureof NKT, CD4⁺, CD8⁺ 0 and CD4⁺CD25⁺ 0 T-cells.

The detection and the quantification of epitope-specific T-cells and thesearch for target epitopes of reactive T-cells can for example beperformed from patients who suffer from an auto-immune disease, achronic inflammatory disease, a microbial infection, a tumour disease,or a transplant rejection, or also from healthy test persons or fromparticipants in therapeutic or preventive studies. In addition, thedetection of epitope-specific T-cells and the search for target epitopesof reactive T-cells can be performed by means of the APC according tothe invention also in primates or other animals which posses T-cells.

For the performance of the method for example blood or anotherAPC-containing and/or T-cell-containing body fluid is extracted from atest person or a patient and the APC are transduced with the genetransfer vectors according to the invention. The transduction of the APCwith the vectors according to the invention can be performed in blood,purified PBMC populations, or in separated APC populations. Theisolation of the purified or of the separated populations can beperformed also after the transduction. The APC according to theinvention which were generated this way, are subsequently incubated withisolated T-cell populations, in particular CD4⁺ T-cells but also CD8⁺T-cells, CD161⁺ NKT cells, CD4⁺CD25⁺ regulatory T-cells, any mixture ofCD4⁺, CD8⁺, CD4⁺CD25⁺ and CD161⁺ NKT cells, or also T-cell-containingcell mixtures, or T-cell-containing body fluid of the same patient for0.5 to 2 hours, 2 to 6 hours or for 6 to 12 hours, or for 12 to 36hours, or for 36 to 168 hours or for more than 168 hours, using suitableconditions for the cultivation, for example 37° C. in a humidifiedatmosphere with 5 to 8% CO₂ in T-cell medium (RPMI 1640 with 2-10%heat-inactivated (30 min., 56° C.) human AB serum, 2 mM glutamine and100 mg/ml kanamycin or gentamycin (all components from PanSystems,Aidenbach, Germany). The incubation can be performed in absence orpresence of suitable (co)-stimulatory substances, like for examplecytokines, mitogens or antibodies. Alternatively, the T-cell-containingcell populations can also be derived from patients/test persons withcompatible MHC-pattern. If reactive T-cells, which recognise APC beingtransduced with the gene transfer vectors and which present specificT-cell epitopes, are present in the mixed APC/T-cell culture, theseinduce or inhibit the marker expression of the sub-populations of theAPC according to the invention. If APC according to the invention, whichpresent T-cell epitopes being recognised by the reactive T-cells of thetest person/patient, are present in the mixed APC/T-cell culture, themarker gene expression is induced or inhibited as a result of theepitope-specific recognition in these APC.

In the further method, the measurably changed (increased or induced)expression of the polypeptides being under the control of the genomiccontrollable promoter in the APC serves in step (d) as a marker for theT-cell recognition, wherein the change is a result of theepitope-specific recognition of the APC according to the invention by aT-cell.

For example, the increased expression of the OX40 ligand (OX40L) and ofthe 4-1 BB ligand (4-1 BBL) (den Haan and Bevan, 2000) as well as of theco-stimulatory proteins B7.1 (CD80), B7.2 (CD86) and of the Fas ligand(FasL) as a result of the epitope-specific recognition in the APC mayserve as markers. In addition, the expression of any polypeptide whichis measurably increased or decreased in APC according to the inventionas a result of the epitope-specific recognition of the APC according tothe invention by a reactive T-cell, is suited as a marker.

The presence and the number of marker-positive APC can for example bedetected by means of FACS, ELISA or the immuno-fluorescence technologyusing specific chemically modified antibodies and pairs of antibodies,respectively. Alternatively, markers with enzymatic functions aredetected by the addition and reaction of a suitable substrate.Epitope-presenting APC according to the invention which show expressionof one of the described markers can be detected, isolated andindividualised by means of different immunological and biochemicalmethods using commercially available devices and test systems. Thus, theexpression of selected markers can be detected for example by means ofFACS, ELISA or immuno-fluorescence technology, for example usingspecific, chemically modified antibodies and pairs of antibodies,respectively. Alternatively, markers with enzymatic functions aredetected by the addition and reaction of suitable substrates. Theisolation and individualisation of marker-positive APC can be performedusing the FACSsort technology (for example by the companies Coulter,Becton Dickinson). The molecular biological and immunological methodswhich are used in the search for epitopes, e.g. the performance ofFACSscan analyses, FACSsorting, ELISA assays, immuno-fluorescenceanalyses, detection of enzymatic reactions, the preparation of total DNAfrom cells, the PCR as well as sequencing of nucleic acid are state ofthe art.

The isolation and characterisation of the polynucleotide encoding thetarget epitopes of reactive T-cells from selected marker-positive APCaccording to the invention can be performed by means of differentmethods which are described exemplarily in the following. For example,the PCR-method is suited for the identification of the target epitopesof reactive T-cells from the selected, marker-positive APC according tothe invention. Total-DNA is prepared from the individualisedmarker-positive APC according to the invention using a commercial kit(for example of the companies Stratagene or Qiagen) and the unknownnucleic acid sequence of the searched T-cell epitope is amplified usingsuitable oligonucleotides (primers) by the PCR-method. The primerspreferentially exhibit constant regions within the nucleic acidsaccording to the invention being located in direct proximity to theidentified polynucleotides.

Alternatively, the lysate of the selected and individualisedmarker-positive APC can be used as a template for the PCR-reaction, too.By using specific primers outside of the polynucleotides which are underthe control of the constitutive promoter (P₂) in the flanking regions onthe non-viral and viral vectors according to the invention, the unknowncDNAs being under the control of the constitutive promoter may beamplified, characterised by sequencing and potentially identified by thecomparison with sequences of a database.

Alternatively, total-DNA can be prepared from the individualised,marker-positive APC or from a mixture of marker-positive APC using e.g.a commercial kit. Subsequently, the obtained DNA is cleaved with arestriction endonuclease which recognition sequence flanks theconstitutive promoter including the polynucleotide being under itscontrol as well as a bacterial resistance and the bacterial origin ofreplication in the 5′ and 3′ region (see FIG. 1B). Subsequently, theobtained DNA fragments are re-ligated and transformed in suitablebacteria, for example DH10B bacteria. If episomal plasmids were used astransfer systems according to the invention for the transduction of theAPC the total DNA obtained from the marker-positive APC can be useddirectly for transformation of suitable bacteria.

Bacteria, which were transformed according to this method with plasmidswhich contain the promoter P₂ according to the invention and thepolynucleotide being under the control of this promoter in combinationwith a bacterial antibiotic resistance and the bacterial origin of areplication, can be selectively cultivated and individualised byspreading on agar plates which contain the respective antibiotic. Aftercultivation of the clonal bacterial colonies in a respectiveantibiotic-containing selective medium the plasmid DNA is obtained fromthe bacteria by means of common methods, for example by alkaline lysis.

The plasmids that have been purified from the bacteria are directly usedas templates for the sequencing reaction. By the use of specific primersthat bind to the conserved regions of the vector according to theinvention close-by the polynucleotide to be identified which is underthe control of the promoter P₂ according to the invention, thepolynucleotides being under the control of the promoter P₂ can bedetermined by sequencing, and their identity can be potentiallydetermined by comparison with sequences in biological and medicaldatabases. For the confirmation of the cDNA-sequences identified bythese methods and for the elimination of false positive results, theobtained nucleic acid sequences are re-cloned under the control of theconstitutive promoter into the vectors according to the invention andtransfected by means of one of the transfer systems according to theinvention in APC. The APC generated this way are then re-tested forrecognition by reactive T-cells of the same patient. By means of aprogressive 5′- and 3′-truncation of the identified polynucleotides andthe insertion of these polynucleotides by means of transfer systemsaccording to the invention into APC the minimal length of the recognisedtarget region can be determined by co-cultivation experiments withreactive T-cells of the patient/test person.

By means of the method according to the invention, for examplepolypeptides and epitopes can be identified which are important in theT-cell mediated recognition and control of diseases which are induced bymicro-organisms and parasites, in the T-cell mediated recognition andcontrol of tumour diseases, and which represent target structures ofmisguided, endogenous T-cells in the case of auto-immune diseases andchronic inflammatory processes or that represent target structures ofendogenous T-cells in the case of the rejection of transplanted tissuesor organs.

The methods according to the invention are suited for a multiplicity ofdifferent medical applications. By means of the method according to theinvention, epitope- and polypeptide-specific T-cells can be identifiedfor the early diagnostics of auto-immune diseases; for monitoring ofreactive T-cells in patients with auto-immune diseases, which show achronic progressive or relapsing/remitting course of disease; fordetermination of the efficiency of therapeutic treatments of diseaseswith T-cells being involved; for screening of the security andefficiency of medicaments that elicit a deletion or anergy of T-cells orcause a general immune suppression; for monitoring of the efficiency oftherapeutic and prophylactic vaccinations for the induction of T-cells;for monitoring and diagnostics of micro-organism- and parasite-induceddiseases with T-cells being involved; for monitoring and diagnostics ofchronic inflammations with T-cells being involved; for monitoring anddiagnostics of tumour antigen-specific T-cells; for monitoring anddiagnostics of T-cells that play a role in the rejection of transplantsor for the targeted selection of test persons for studies invaccinations and the testing of therapeutic treatments.

Furthermore, the invention comprises a kit for a diagnostic detection ofauto-reactive T-cells, comprising suitable vectors or transfer systemsaccording to the invention for the generation of APC according to theinvention, packed in suitable containers; comprising a detailedinstruction for the performance of the diagnostic detection and asuitable container for transport and storage of the kit according to theinvention.

The present invention results in the following advantages for the searchfor epitopes. The use of the vectors according to the invention incombination with gene libraries facilitates the quick identification ofT-cell epitopes from presently known and characterised polypeptides butalso from presently not yet described and characterised, respectively,polypeptides. In addition, the use of gene libraries in one or a verylimited number of experimental approaches many target epitopes ofreactive T-cells of a test person/patient can be isolated and describedsimultaneously. Therefore, this method in comparison to the as yet usedmethods for the search for epitopes accounts for a significantly reducedexpense of time and costs.

Poly-specific T-cells of a patient may be used for the search forepitopes, thereby avoiding the very time and labour consuming step forthe generation of T-cell lines. APC expressing their target epitopes ofreactive T-cells can be selected very quickly because of the markerexpression by commercially available devices (for example FACSsort) andthe genes of the searched T-cell epitopes may be identified by means ofmolecular biological routine methods.

The method according to the invention is universally suited for thesearch for epitopes in different test persons/patients. Gene transfermeans carrying the vectors according to the invention can be useduniversally for the genetic manipulation of the APC for the search forepitopes independently of the haplotype in all test persons/patients.The method according to the invention is suited for the search fortarget epitopes of different T-cell populations (especially of CD4⁺T-helper cells but also of CD8⁺ cytotoxic T-cells, CD161⁺NKT-cells andCD4⁺CD25⁺ regulatory T-cells).

The present invention results in the following advantages in the case ofthe detection and diagnostics of epitope-specific T-cells. An essentialadvantages of the method according to the invention resides in the factthat the method is based on the measurement of a marker gene expressionwhich is induced by an epitope-specific recognition by a T-cell in APCand that is not based, like in the case of all previous methods, on themeasurement of reactions of the T-cell resulting from a recognition ofthe epitope on the APC. Thus, the sensitivity of the assay system isconsiderably increased. The method of detection for the reactive T-cellsis in comparison to the common methods of diagnostics (CTL-assay,ELISPOT, cytokin-ELISA, proliferation assay) all-purpose, easier tomanage, significantly more cost-effective, less time consuming andsensitive. The presence and number of reactive T-cells can easily bedetected and quantified by means of commercially available and in manydiagnostics laboratories routinely used devices (FACS). The method fordiagnostics according to the invention can be universally usedindependently of the haplotype of the test person/patient for thedetection of the reactive T-cells. The method according to the inventionis suited for the detection of various epitope-specific T-cellpopulations (especially CD4⁺ T-helper cells but also CD8⁺ cytotoxicT-cells, CD161⁺ NKT-cells and CD4⁺CD25⁺ regulatory T-cells).

The following examples illustrate the present invention:

EXAMPLE 1 Cloning of the Vector Backbone According to the Invention forthe Expression of Polypeptides Under the Control of a Promoter which isConstitutively Active in APC (P₂)

As starting vectors for the cloning of the vector backbones according tothe invention served the plasmid pcDNA3.1(+) (Invitrogen) containing5446 base pairs (bp) as well as the vector pcDNA3.1(+)dNeo which wasgenerated by the deletion of a 1905 bp-comprising DNA-fragment includingthe coding region for neomycin (bp 1289-3194) from the plasmidpcDNA3.1(+). Therefore the nucleic acid sequence of thepcDNA3.1(+)dNeo-plasmid was obtained by means of the polymerase chainreaction (PCR) using the following conditions for amplification:introductory denaturation step: 95° C., 1 min., followed by a 3-step PCR(35 cycles) with the following conditions: denaturation: 94° C., 1 min.,annealing 55° C., 1 min., elongation: 72° C., 3.5 min. with a finalpolymerisation step at 72° C. for 10 min. with subsequent permanentcooling at 4° C. For the amplification the primers P1(5′-GCTGGTTCTTTCCGCCTCAGAAGC-3′; SEQ ID NO: 6) and P2(5′-CACTGCATTCTAGTTGTGGTTTG-3′; SEQ ID NO: 7) were used. The obtainedPCR-product was purified by means of a QIAquick PCR Purification Kit(Qiagen) following the manufacturer's protocol, phosphorylated andfinally re-ligated. The obtained plasmid comprising 3541 bp was denotedpcDNA3.1(+)dNeo.

For the insertion of different control sequences being constitutivelyactive in APC, these sequences were amplified by PCR from suitablematerial (chromosomal DNA of human and murine origin) and inserted intothe vector backbones pcDNA3.1(+) and pcDNA3.1(+)dNeo by means ofsuitable restriction sites.

Amplification of the Murine PGK Promoter

The 621 bp comprising sequence of the murine PGK promoter was obtainedby PCR from isolated chromosomal DNA of the murine cell line C2C12 (ATCCnumber CRL-1772) using the following conditions: introductorydenaturation step: 95° C., 2 min., followed by a 3-step PCR (35 cycles)with the following conditions: denaturation: 95° C., 45 sec., annealing66° C., 1 min., elongation: 72° C., 1.5 min., with a finalpolymerisation step at 72° C. for 10 min. with final permanent coolingat 4° C. For the amplification the primers P3 (5′ primer:5′-GCAGGCTCGCGACTACCGGGTAGGGGAGGCGC-3′; SEQ ID NO: 8) and P4 (3′ primer:5′-GCAGGCGGATCCACGCGCTTCTACAAGGCGCTTGC-3′; SEQ ID NO: 9) were used.

Amplification of the Human PKG Promoter

The 531 bp comprising sequence of the human PGK promoter was isolatedvia PCR from isolated chromosomal DNA from human PBMCs using thefollowing conditions for amplification: introductory denaturation step:98° C., 2 min., followed by two subsequent 3-step PCRs with thefollowing conditions: first round of PCR-amplification (15 cycles):denaturation: 95° C., 1 min., annealing 58° C., 1 min., elongation: 72°C., 1.5 min. Subsequently a second round of PCR-amplification followed(25 cycles) with the following conditions: denaturation: 95° C., 1 min.,annealing 69° C., 1 min., elongation: 72° C., 1.5 min. with a finalpolymerisation step at 72° C. for 10 min. with subsequent permanentcooling at 4° C. For the amplification the primers P5 (5′ primer:5′-GCAGGCTCGCGACGGGGTTGGGGTTGCGCC-3′; SEQ ID NO: 10) and P6 (3′ primer:5′-GCAGGCGGATCCTTTGGAAATACAGCTGGGGAG-3′; SEQ ID NO: 11) were used.

Amplification of the Human DHFR Promoter

The 479 bp comprising sequence of the human dihydrofolate-reductase(DHFR) promoter was obtained via PCR from isolated chromosomal DNAderived from human PBMCs using the following conditions foramplification: introductory denaturation step: 95° C., 2 min.,subsequently followed by a 3-step PCR (30 cycles) with the followingconditions: denaturation: 95° C., 45 sec., annealing 71° C., 1 min.,elongation: 72° C., 1.5 min., with a final polymerisation step 72° C.for 10 min. with a subsequent permanent cooling at 4° C. For theamplification the primers P7 (5′ primer:5′-GCAGGCTCGCGACGATGGCCCTGCCCAGTCCC-3′; SEQ ID NO: 12) and P8 (3′primer: 5′-GCAGGCGGATCCGACAGCAGCGGGAGGACCTC-3′; SEQ ID NO: 13) wereused.

Amplification of Human EF-1α Promoter

Hereby the 1244 bp comprising sequence of the human EF-1α promoter wasobtained by PCR from isolated chromosomal DNA derived from human PBMCsusing the following conditions for amplification: introductorydenaturation step: 98° C., 2 min., followed by two subsequent 3-stepPCRs with the following conditions: first round of PCR-amplification (15cycles) denaturation: 95° C., 1 min., annealing 58° C., 1 min.,elongation: 72° C., 10 min. Subsequently a second round ofPCR-amplification followed (30 cycles) with the following conditions:denaturation: 98° C., 1 min., annealing 67° C., 1 min., elongation: 72°C., 2 min. with a final polymerisation step at 72° C. for 10 min. withsubsequent permanent cooling at 4° C. For the amplification the primersP9 (5′ primer: 5′-GCAGGCTCGCGAGGCTCCGGTGCCCGTCAGTG-3′; SEQ ID NO: 14)and P10 (3′ primer: 5′-GCAGGCGGATCCACCTAGCCAGCTTGGGTCTCC-3′; SEQ ID NO:15) were used.

Amplification of the Human Ubiquitin C (UbC) Promoter

The 1210 bp comprising sequence of the human UbC promoter was amplifiedby PCR from isolated chromosomal DNA derived from human PBMCs using thefollowing conditions: introductory denaturation step: 98° C., 2 min.,followed by two subsequent 3-step PCRs with the following conditions:first round of PCR-amplification (15 cycles) denaturation: 95° C., 50sec., annealing 58° C., 45 sec., elongation: 72° C., 3 min. Subsequentlya second round of PCR-amplification followed (25 cycles) with thefollowing conditions: denaturation: 95° C., 50 sec., annealing 71° C.,45 sec., elongation: 72° C., 3 min., with a final polymerisation step at72° C. for 10 min. with a subsequent permanent cooling at 4° C. For theamplification the primers P11 (5′ primer:5′-GCAGGCTCGCGAGGCCTCCGCGCCGGGTTTTGG-3′; SEQ ID NO: 16) and P12 (3′primer: 5′-GCAGGCGGATCCGTCTAACAAAAAAGCCAAAAACG-3′; SEQ ID NO: 17) wereused.

Amplification of the Human ICI Promoter

The 569 bp comprising sequence of the human ICI promoter was amplifiedby PCR from isolated chromosomal DNA derived from human PBMCs using thefollowing conditions: introductory denaturation step: 95° C., 2 min.,followed by two subsequent 3-step PCRs with the following conditions:first round of PCR-amplification (15 cycles): denaturation: 95° C., 45sec., annealing 58° C., 1 min., elongation: 72° C., 1.5 min.Subsequently followed a second round of PCR-amplification (25 cycles)with the following conditions: denaturation: 95° C., 45 sec., annealing66° C., 1 min., elongation: 72° C., 1.5 min. with a final polymerisationstep at 72° C. for 10 min. with subsequent permanent cooling at 4° C.For the amplification the primers P13 (5′ primer:5′-GCAGGCTCGCGAGCTGTAATTTCTAATCTAAACC-3′; SEQ ID NO: 18) and P14 (3′primer: 5′-GCAGGCGGATCCAGCAGCAGAGTGCGGCAACAC-3′; SEQ ID NO: 19) wereused.

The restriction sites (NruI in the 5′ primer and BamHI in the 3′primer), which are present in the primers being used for amplificationof the above mentioned promoters, are each depicted in italics.

The preparation of human PBMC was performed as described in thefollowing: freshly extracted whole blood of a healthy test person wasspiked with 20 IU/ml heparin. 50 ml-Leukosep-vials with a separatingmembrane were filled with 15 ml Ficoll-Histopaque (Sigma, Deisenhofen,Germany) and centrifuged for 2 min. at 500×g. The tubes were filled withthe heparinised whole blood and diluted 1:1 with sterile PBS/0.5% BSA.The samples were centrifuged for 30-40 min. at 400×g withoutdeceleration of the rotor at the end of the centrifugation. Thelymphocytes-containing, turbid interface was carefully removed andwashed in 3 volumes of T-cell medium (RPMI 1640 medium with 10% heatinactivated (30 min. at 56° C.) human AB serum, 2 mM glutamine and 100mg/ml kanamycin and gentamycin, respectively (all components fromPanSystems, Aidenbach, Germany)) for three times.

The isolation of genomic DNA from human PBMC or murine C2C12 musclecells was performed with the “DNA extraction kit” (Stratagene) followingthe manufacturer's protocol. For this the cells were washed 2× with coldPBS (phosphate buffered saline) and the cell number was determined.1×10⁸ cells each were transferred to 15 ml plastic tubes and put on iceimmediately. All subsequent working steps were performed on ice, too.After a centrifugation for 15 minutes (350×g, 4° C.) the cells werere-suspended in 11 ml solution 2 (50 mM Tris/HCl, pH 8.0, 20 mM EDTA, 2%SDS) and homogenised with a Dounce homogeniser. After this Pronase(Stratagene) was added to a final concentration of 100 μg/ml and thesample was incubated overnight at 37° C., slightly shaking.Subsequently, the sample was cooled on ice for 10 min. and 4 ml of anice-cold solution 3 (saturated NaCl-solution) were added. After shortmixing of the solutions a further incubation on ice took place for 5min. and the pellet was centrifuged at 4° C. (15 min., 2000×g). Afterthis, the supernatant was transferred into a sterile 50 mlcentrifugation tube by a cut plastic pipette, RNAse was added to a finalconcentration of 20 μg/ml and the mixture was incubated for 15 min. at37° C. After addition of two volumes of 100% ethanol and slight shakingthe precipitation of the genomic DNA in a white smear could bevisualised. The DNA could now be either wound up on a glass rod or bepelleted by a centrifugation for 15 minutes at 4° C. and 2000×g. After awashing step with 2 ml of cold 70% ethanol the DNA was transferred to anEppendorf reaction tube, centrifuged and air-dried for 15 min. at roomtemperature. The DNA pellet was re-suspended in H₂O_(bid.) and dissolvedovernight at room temperature and for 1-2 hours at 55° C., respectively.The determination of the DNA concentration was performed photometricallyat 258 mn.

The obtained PCR-products were purified by means of a QIAquick PCRpurification kit (Qiagen) following the manufacturer's protocol andsubsequently 20 μg of the respective purified PCR-bands were digested in100 μl with the restriction enzymes NruI and BamHI. At the same time 20μg each of the vector backbones pcDNA3.1(+) and pcDNA3.1 (+)dNeo weredigested with the same restriction enzymes (NruI and BamHI). Theobtained restriction products were separated by a 1% agarose gel, thepolynucleotides for the linearised vector backbones and the differentpromoters were cut out of the gel and the nucleic acids were purified bymeans of a QIAquick gel extraction kit (Qiagen). Subsequently, theobtained polynucleotides of the vector backbones were ligated with thedifferent digested PCR-amplificates with the respective promotersequences overnight at 16° C., and the ligation samples weresubsequently transformed into bacteria (DH5α). After an incubation for 1hour at 37° C. in LB medium without a selection antibiotic thetransformed bacteria were spread on LB_(Amp) plates and incubatedovernight at 37° C. The obtained bacterial colonies were then cultivatedin LB_(Amp) medium, the bacterial plasmid-DNA was isolated by thetechnique of the alkaline lysis and characterised by a restrictiondigest using the restriction endonucleases BglII/EcoRI. The vectorsgenerated this way were denoted pcDNA3.1(+)mPGK, pcDNA3.1(+)hPGK,pcDNA3.1(+)hDHFR, pcDNA3.1(+)hEF-1α, pcDNA3.1(+)hUbC, pcDNA3.1(+)hICI,and pcDNA3.1(+)dNeomPGK, pcDNA3.1(+)dNeohPGK, pcDNA3.1(+)dNeohDHFR,pcDNA3.1(+)dNeohEF-1α, pcDNA3.1(+)dNeohUbC and pcDNA3.1(+)dNeohICI,respectively.

The 224 bp comprising bovine growth hormone (BGH) polyadenylationsequence was amplified from the pcDNA3.1(+)-vector by PCR using suitablePCR conditions (introductory denaturation step: 95° C., 2 min.,subsequent a 3-step PCR (35 cycles) with the following conditions:denaturation: 95° C., 30 sec.; annealing 55° C., 1 min.; elongation: 72°C., 1 min., and a final polymerisation step at 72° C. for 10 min. with asubsequent permanent cooling at 4° C.). For amplification the primer P15(5′ primer: 5′-GGCGGGGGATCCCTGTGCCTTCTAGTTGCC-3′; SEQ ID NO: 20) and P16(3′ primer: 5′-GGCGGGAGATCTCCATAGAGCCCACCGC-3′; SEQ ID NO: 21) wereused. The restriction sites contained within the primers (BamHI in the5′ primer; BglII in the 3′ primer) are depicted in italics. ThePCR-amplified band was subsequently purified as described previously,restricted with the restriction enzymes BglII and BamHI and the in sucha way obtained band was ligated into the BamHI-linearised plasmidspcDNA3.1(+)mPGK, pcDNA3.1(+)hPGK, pcDNA3.1 (+)hDHFR, pcDNA3.1(+)hEF-1α,pcDNA3.1(+)hUbC, pcDNA3.1(+)hICI, and pcDNA3.1(+)dNeomPGK,pcDNA3.1(+)dNeohPGK, pcDNA3.1(+)dNeohDHFR, pcDNA3.1(+)dNeohEF-1α,pcDNA3.1(+)dNeohUbC, respectively, and pcDNA3.1(+)dNeohICI. The in sucha way generated vectors received the denotations pcDNA3.1(+)mPGKPA,pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA, pcDNA3.1(+)hEF-1αPA,pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, and pcDNA3.1(+)dNeomPGKPA,pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA, pcDNA3.1(+)dNeohEF-1αPA,pcDNA3.1(+)dNeohUbCPA, respectively, and pcDNA3.1(+)dNeohICIPA.

However, the vector backbones pcDNA3.1(+) (Invitrogen) as well as thevector pcDNA3.1(+)dNeo are analogously suited as vectors for theexpression of any polypeptides in APC. They contain the CMV promoterbeing constitutively active in APC in connection with the bovine growthhormone (BGH) polyadenylation sequence.

Subsequently, polynucleotides encoding polypeptides like for example theEpstein-Barr virus BZLF-1 protein, the human myelin basic protein (MBP)and the HIV-1 p24 capsid protein, were cloned under the control of theabove mentioned promoter being constitutively active in APC. For thisthe desired polynucleotides were amplified by PCR, the PCR-amplificatespurified, restricted with suitable restriction endonucleases andinserted into vectors which have been digested with the respectiverestriction endonucleases.

Generation of Plasmids for the Constitutive Expression of theEpstein-Barr Virus BZLF-1 Protein

The cDNA for the EBV BZLF-1 protein was amplified from viral DNA of theEBV strain B95-8 from total DNA of infected marmoset cells by means ofPCR using suitable PCR conditions (introductory denaturation step: 95°C., 2 min., subsequently followed by a 3-step PCR (15 cycles) with thefollowing conditions: denaturation: 95° C., 30 sec.; annealing 52° C., 1min.; elongation: 72° C., 2 min., after this another PCR (25 cycles)took place with the following conditions: denaturation: 95° C., 30 sec.;annealing 63° C., 1 min.; elongation: 72° C., 2 min. and a finalpolymerisation step at 72° C. for 10 min. with subsequent permanentcooling at 4° C.). In the case of the PCR-reaction for the amplificationof the BZLF-1 cDNA 2.5% DMSO were added to the PCR sample. Foramplification the primers P17 (5′ primer:5′-GGCGGAGATCTTTAGAAATTTAAGAGATCC-3′; SEQ ID NO: 22) and P18 (3′ primer:5′-GGCGGGAGATCTATGATGGACCCAAACTCG-3′; SEQ ID NO: 23) were used. Theamplified 970 nucleotides comprising band which was amplified by PCR waspurified as described previously, restricted with the restriction enzymeBglII and the obtained band was ligated into the BamHI-linearisedexpression vectors pcDNA3.1(+)mPGKPA, pcDNA3.1(+)hPGKPA,pcDNA3.1(+)hDHFRPA, pcDNA3.1(+)hEF-1αPA, pcDNA3.1(+hUbCPA,pcDNA3.1(+)hICIPA, respectively into pcDNA3.1(+)dNeomPGKPA,pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA, pcDNA3.1(+)dNeohEF-1αPA,pcDNA3.1(+)dNeohUbCPA, pcDNA3.1(+)dNeohICIPA, pcDNA3.1(+) and intopcDNA3.1(+)dNeo. The expression vectors generated this way obtained thedenotation pcDNA3.1(+)mPGKPA-Z, pcDNA3.1(+)hPGKPA-Z,pcDNA3.1(+)hDHFRPA-Z, pcDNA3.1(+)hEF-1αPA-Z, pcDNA3.1(+)hUbCPA-Z,pcDNA3.1(+)hICIPA-Z, respectively pcDNA3.1(+)dNeomPGKPA-Z,pcDNA3.1(+)dNeohPGKPA-Z, pcDNA3.1(+)dNeohDHFRPA-Z,pcDNA3.1(+)dNeohEF-1αPA-Z, pcDNA3.1(+)dNeohUbCPA-Z,pcDNA3.1(+)dNeohICIPA-Z, pcDNA3.1(+)-Z (FIG. 2) and pcDNA3.1(+)dNeo-Z.

Generation of Plasmids for the Constitutive Expression of the HumanMBP-Protein

The cDNA for the human MBP-protein was amplified from total DNA frombrain cells by means of PCR using suitable conditions (introductorydenaturation step: 95° C., 2 min., subsequently followed by a 3-step PCR(35 cycles) with the following conditions: denaturation: 95° C., 30sec.; annealing 55° C., 1 min.; elongation: 72° C., 2 min. and the finalpolymerisation step at 72° C. for 10 min. with subsequent permanentcooling at 4° C.). For amplification the primers P19 (5′ primer:5′-GGCGGAGATCTATGGCGTCACAGAAGAGACC-3′; SEQ ID NO: 24) and P20 (3′primer: 5′-GGCGGGAGATCTTCAGCGTCTAGCCATGGG-3′; SEQ ID NO: 25) were used.The 585 nucleotides comprising band which was amplified by PCR was thenpurified as described previously, restricted with the restriction enzymeBglII and the obtained band was ligated into the BamHI-linearisedplasmids pcDNA3.1(+)mPGKPA, pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA,pcDNA3.1(+)hEF-1αPA, pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, respectivelyinto pcDNA3.1(+)dNeomPGKPA, pcDNA3.1(+)dNeohPGKPA,pcDNA3.1(+)dNeohDHFRPA, pcDNA3.1(+)dNeohEF-1αPA, pcDNA3.1(+)dNeohUbCPA,pcDNA3.1(+)dNeohICIPA, pcDNA3.1(+) and pcDNA3.1(+)dNeo. The expressionvectors generated this way obtained the denotationspcDNA3.1(+)mPGKPA-MBP, pcDNA3.1(+)hPGKPA-MBP, pcDNA3.1(+)hDHFRPA-MBP,pcDNA3.1(+)hEF-1αPA-MBP, pcDNA3.1(+)hUbCPA-MBP, pcDNA3.1(+)hICIPA-MBP,respectively pcDNA3.1(+)dNeomPGKPA-MBP, pcDNA3.1(+)dNeohPGKPA-MBP,pcDNA3.1(+)dNeohDHFRPA-MBP, pcDNA3.1(+)dNeohEF-1αPA-MBP,pcDNA3.1(+)dNeohUbCPA-MBP, pcDNA3.1(+)dNeohICIPA-MBP, pcDNA3.1(+)-MBPand pcDNA3.1(+)dNeo-MBP.

A codon-optimised DNA for the HIV-1 p24 capsid protein was amplifiedfrom a synthetically produced nucleic acid according to a publishedHIV-1 gag sequence (Graf et al. (2000), J. Virol. 74:10822) by means ofPCR using suitable conditions (introductory denaturation step: 95° C., 2min., subsequently followed by a 3-step PCR (30 cycles) with thefollowing conditions: denaturation: 95° C., 45 sec.; annealing 66° C., 1min.; elongation: 72° C., 2 min. and a final polymerisation step at 72°C. for 10 min. with subsequent permanent cooling at 4° C.).Codon-optimised genes can be obtained from several companies (forexample GeneArt, Regensburg, Germany; Entelechon, Regensburg, Germany).For amplification the primers P21 (5′ primer:5′-GGCGGTCTAGAGCCGCCACCATGCCCATCGTG-3′; SEQ ID NO: 26) and P22 (3′primer: 5′-GGCGGGGAATTCTCACAGCAGCCTGGCCTTGTG-3′; SEQ ID NO: 27) wereused. The obtained PCR product was subsequently purified as describedpreviously, restricted with the restriction enzyme BglII and theobtained band was ligated into the BamHI-linearised plasmidspcDNA3.1(+)mPGKPA, pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA,pcDNA3.1(+)hEF-1αPA, pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, respectivelypcDNA3.1(+)dNeomPGKPA, pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA,pcDNA3.1(+)dNeohEF-1αPA, pcDNA3.1(+)dNeohUbCPA, pcDNA3.1(+)dNeohICIPA,pcDNA3.1(+) and pcDNA3.1(+)dNeo. The expression vectors generated thisway obtained the denotations pcDNA3.1(+)mPGKPA-p24,pcDNA3.1(+)hPGKPA-p24, pcDNA3.1(+)hDHFRPA-p24, pcDNA3.1(+)hEF-1αPA-p24,pcDNA3.1(+)hUbCPA-p24, pcDNA3.1(+)hICIPA-p24, respectivelypcDNA3.1(+)dNeomPGKPA-p24, pcDNA3.1 (+)dNeohPGKPA-p24,pcDNA3.1(+)dNeohDHFRPA-p24, pcDNA3.1(+)dNeohEF-1αPA-p24,pcDNA3.1(+)dNeohUbCPA-p24, pcDNA3.1(+)dNeohICIPA-p24, pcDNA3.1(+)-p24and pcDNA3.1(+)dNeo-p24.

Generation of Plasmids for the Constitutive Expression of the “EnhancedGreen Fluorescent Protein” (eGFP)

The gene for the “enhanced green fluorescent protein” (eGFP) wasamplified from the plasmid pRC/CMV-EGFP (Vogel et al., 1998,BioTechniques 143, 1967-1983) by means of PCR using suitable PCRconditions (introductory denaturation step: 95° C., 2 min., subsequentlya 3-step PCR (35 cycles) with the following conditions: denaturation:95° C., 30 sec.; annealing 55° C., 1 min.; elongation: 72° C., 2 min.and a final polymerisation step at 72° C. for 10 min. with subsequentpermanent cooling at 4° C. For amplification the primers P23 (5′ primer:5′-GGCGGGAGATCTCGCCACCATGGTGAGCAAGG-3′; SEQ ID NO: 28) and P24 (3′primer: 5′-GGCGGGAGATCTTTACTTGTACAGCTCGTCC-3′; SEQ ID NO: 29) were used.The PCR-amplified specific band having the size of 752 bp wassubsequently purified as described previously, restricted with therestriction enzyme BglII, and the obtained band was ligated into theBamHI-linearised expression vectors pcDNA3.1(+)mpGKPA,pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA, pcDNA3.1(+)hEF-1αPA,pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, respectivelypcDNA3.1(+)dNeomPGKPA, pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA,pcDNA3.1(+)dNeohEF-1αPA, pcDNA3.1(+)dNeohUbCPA, pcDNA3.1(+)dNeohICIPA,pcDNA3.1(+) and into pcDNA3.1(+)dNeo. The expression vectors generatedthis way obtained the denotations pcDNA3.1(+)mPGKPA-eGFP,pcDNA3.1(+)hPGKPA-eGFP, pcDNA3.1(+)hDHFRPA-eGFP,pcDNA3.1(+)hEF-1αPA-eGFP, pcDNA3.1(+)hUbCPA-eGFP,pcDNA3.1(+)hICIPA-eGFP, respectively pcDNA3.1(+)dNeomPGKPA-eGFP,pcDNA3.1(+)dNeohPGKPA-eGFP, pcDNA3.1(+)dNeohDHFRPA-eGFP,pcDNA3.1(+)dNeohEF-1αPA-eGFP, pcDNA3.1(+)dNeohUbCPA-eGFP,pcDNA3.1(+)dNeohICIPA-eGFP, pcDNA3.1(+)-eGFP and pcDNA3.1(+)dNeo-eGFP.

EXAMPLE 2 Generation of Vectors which Facilitate a Targeted Transport ofthe Polypeptides Being Expressed by Means of the Constitutive Promoter(P₂) into the Endolysosome

For the enhancement of the endo/lysosomal degradation of thepolypeptides being expressed by the means of the constitutive promoter(P₂), additionally different lysosomal targeting signals, for examplethe cytoplasmatic region of the Ii chain, or regions of the “invariantchain”, or of LAMP-1, LAMP-2, LIMP-1 (CD63), LAP or MHC-class IIproteins were introduced into the plasmids pcDNA3.1(+)mPGKPA,pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA, pcDNA3.1(+)hEF-1αPA,pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, respectively intopcDNA3.1(+)dNeomPGKPA, pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA,pcDNA3.1(+)dNeohEF-1αPA, pcDNA3.1(+)dNeohUbCPA, pcDNA3.1(+)dNeohICIPA,pcDNA3.1(+) and into pcDNA3.1(+)dNeo.

Construction of Expression Vectors which Express Chimeric Proteins ofSignalling Peptide/Polypeptide/LAMP-1

The cloning of chimeric polypeptides being composed of the N-terminalsignalling peptide of the human LAMP-1 protein, any arbitrarypolypeptide, for example the EBV BZLF-1 protein or the human MBP proteinas well as the transmembrane domain and the cytoplasmatic part of LAMP-1was performed sequentially using the compatible restriction sites BamHIand BglII.

The nucleic acid sequence encoding the signalling peptide of LAMP-1 wasamplified from isolated chromosomal DNA derived from human PBMCs bymeans of PCR using a high fidelity Pfu polymerase (Stratagene) and thefollowing conditions: introductory denaturation step: 95° C., 2 min.,subsequently followed by a 3-step PCR (35 cycles) with the followingconditions: denaturation: 95° C., 1 min.; annealing 60° C., 1 min.;elongation: 72° C., 1 min.; final polymerisation step at 72° C. for 10min. with subsequent permanent cooling at 4° C. For amplification theprimers P25 (5′ primer: 5′-GCAGGAGATCTTATGGCGCCCCGC-3′; SEQ ID NO: 30)and P26 (3′ primer: 5′-GCAGGCGGATCCTCAAAGAGTGCTGA-3′; SEQ ID NO: 31)were used.

The Amplification of Polynucleotides Encoding the BZLF-1 and MBPProteins Using the Amplification Conditions which were DescribedPreviously

For amplification of the BZLF-1 gene the primers P27 (5′ primer:5′-GGCGGGGATCCTTAGAAATTTAAGAGATCC-3′; SEQ ID NO: 32) and primer P28 (3′Primer: 5′-GGCGGGAGATCTATGATGGACCCAAACTCG-3′; SEQ ID NO: 33) were used.

For amplification of the human MBP protein the primers P29 (5′ primer:5′-GGCGGAGATCTATGGCGTCACAGAAGAGACC-3′; SEQ ID NO: 34) and P30 (3′primer: 5′-GGCGGGGGATCCTCAGCGTCTAGCCATGGG-3′; SEQ ID NO: 35) were used.

The amplification of the transmembrane region and cytoplasmatic domainof LAMP-1 was performed by means of PCR using a high fidelity Pfupolymerase (Stratagene) from isolated chromosomal DNA derived from humanPBMCs using the following conditions: introductory denaturation step:95° C., 2 min., subsequently followed by a 3-step PCR (35 cycles) withthe following conditions: denaturation: 95° C., 1 min.; annealing 60°C., 1 min.; elongation: 72° C., 1.5 min., final polymerisation step at72° C. for 10 min. with subsequent permanent cooling at 4° C. Foramplification the primers P31 (5′ primer:5′-GCAGGAGATCTAACAGCACGCTGATC-3′; SEQ ID NO: 36) and P32 (3′ primer:5′-GCAGGCAGATCTCTAGATAGTCTGGTA-3′; SEQ ID NO: 37) were used.

These three components of the nucleic acid encoding the chimericsignalling peptide/polypeptide/LAMP-1 polypeptide fragment were eachrestricted with the respective restriction endonucleases after the PCRreaction, purified and subsequently introduced in three steps into the,in the following denoted, BamHI-restricted expression vectors. In thecase of this method, subsequently the coding regions of the LAMP-1leader, any arbitrary polynucleotide, here for example of the EBV-BZLF-1protein or of the human MBP protein as well as the coding nucleic acidof the C-terminal, the transmembrane domain and the cytoplasmatic partof the LAMP-1 protein were inserted into the plasmids pcDNA3.1(+)mPGKPA,pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA, pcDNA3.1(+)hEF-1αPA,pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, respectively into thepcDNA3.1(+)dNeomPGKPA, pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA,pcDNA3.1(+)dNeohEF-1αPA, pcDNA3.1(+)dNeohUbCPA, pcDNA3.1(+)dNeohICIPA,pcDNA3.1(+) and into pcDNA3.1(+)dNeo. The expression vectors generatedin such a way obtained the denotations (pcDNA3.1(+)mPGKPA-Z/LAMP,pcDNA3.1(+)hPGKPA-Z/LAMP, pcDNA3.1(+)hDHFRPA-Z/LAMP,pcDNA3.1(+)hEF-1PA-Z/LAMP, pcDNA3.1(+)hUbCPA-Z/LAMP,pcDNA3.1(+)hICIPA-Z/LAMP, as well as pcDNA3.1(+)dNeomPGKPA-Z/LAMP,pcDNA3.1(+)dNeohPGKPA-Z/LAMP, pcDNA3.1(+)dNeohDHFRPA-Z/LAMP,pcDNA3.1(+)dNeohEF-1αPA-Z/LAMP, pcDNA3.1(+)dNeohUbCPA-Z/LAMP,pcDNA3.1(+)dNeohICIPA-Z/LAMP, pcDNA3.1(+)-Z/LAMP (FIG. 3) andpcDNA3.1(+)dNeo-Z/LAMP, respectively (pcDNA3.1(+)mPGKPA-MBP/LAMP,pcDNA3.1(+)hPGKPA-MBP/LAMP, pcDNA3.1(+)hDHFRPA-MBP/LAMP,pcDNA3.1(+)hEF-1αPA-MBP/LAMP, pcDNA3.1(+)hUbCPA-MBP/LAMP,pcDNA3.1(+)hICIPA-MBP/LAMP, as well as pcDNA3.1(+)dNeomPGKPA-MBP/LAMP,pcDNA3.1(+)dNeohPGKPA-MBP/LAMP, pcDNA3.1(+)dNeohDHFRPA-MBP/LAMP,pcDNA3.1(+)dNeohEF-1αPA-MBP/LAMP, pcDNA3.1(+)dNeohUbCPA-MBP/LAMP,pcDNA3.1(+)dNeohICIPA-MBP/LAMP, pcDNA3.1(+)-MBP/LAMP andpcDNA3.1(+)dNeo-MBP/LAMP.

EXAMPLE 3 Generation of Vectors which Induce a Membrane Anchoring of thePolypeptide Being Expressed by Means of the Constitutive Promoter (P₂)

The cloning of chimeric polypeptides consisting of the N-terminalsignalling peptide of the human LAMP-1 protein, any arbitrarypolypeptide, for example the EBV BZLF-1 protein or the human MBP proteinas well as of a heterologous transmembrane domain, for example of theEpstein-Barr virus gp220/350 coat protein, was performed sequentiallyusing the compatible restriction sites BamHI and BglII.

The amplification of the nucleic acid sequence encoding the signallingpeptide of LAMP-1 as well as of the coding sequences of the BZLF-1 andMBP proteins was performed as described in detail in example 2.

The amplification of the EBV gp220/350 transmembrane domain (EBV-TM) wasperformed by means of PCR using a high fidelity Pfu polymerase(Stratagene) from the plasmid pBRBamHI-L (Skare and Strominger, (1980),Proc. Natl. Acad. Sci. USA 77, 3860-3864) using the followingconditions: introductory denaturation step: 95° C., 2 min., subsequentlyfollowed by a 3-step PCR (35 cycles) with the following conditions:denaturation: 95° C., 1 min.; annealing 60° C., 1 min.; elongation: 72°C., 1 min., final polymerisation step at 72° C. for 10 min. withsubsequent permanent cooling at 4° C. For amplification the primers P33(5′ primer: 5′-GCAGGAGATCTAGCGGGGCAGGATCCATGCTAGTACTTCAATGGGCCTCTCTG-3′;SEQ ID NO: 38) and P34 (3′ primer:5′-GCAGGCAGATCTTTATACATACCTCTCGGCCTC-3′; SEQ ID NO: 39) were used.

These three components of the nucleic acid encoding the chimericsignalling peptide/polypeptide/EBV-TM polypeptide fragment were eachrestricted by the respective restriction endonucleases after the PCRreaction, purified and inserted sequentially in three steps intoBamHI-restricted, in the following denoted, expression vectors each. Inthe case of this method sequentially the coding regions of the LAMP-1leader, of any arbitrary polynucleotide, here for example of theEBV-BZLF-1 protein or the human MBP protein as well as the nucleic acidencoding the EBV gp220/350 transmembrane domain were ligated into theplasmids pcDNA3.1(+)mPGKPA, pcDNA3.1(+)hPGKPA, pcDNA3.1(+)hDHFRPA,pcDNA3.1(+)hEF-1αPA, pcDNA3.1(+)hUbCPA, pcDNA3.1(+)hICIPA, respectivelypcDNA3.1(+)dNeomPGKPA, pcDNA3.1(+)dNeohPGKPA, pcDNA3.1(+)dNeohDHFRPA,pcDNA3.1(+)dNeohEF-1αPA, pcDNA3.1(+)dNeohUbCPA, pcDNA3.1(+)dNeohICIPA,pcDNA3.1(+) and into pcDNA3.1(+)dNeo. The expression vectors which weregenerated in such a way obtained the denotations(pcDNA3.1(+)mPGKPA-Z/TM, pcDNA3.1(+)hPGKPA-Z/TM,pcDNA3.1(+)hDHFRPA-Z/TM, pcDNA3.1(+)hEF-1αPA-Z/TM,pcDNA3.1(+)hUbCPA-Z/TM, pcDNA3.1(+)hICIPA-Z/TM, as well aspcDNA3.1(+)dNeomPGKPA-Z/TM, pcDNA3.1(+)dNeohPGKPA-Z/TM,pcDNA3.1(+)dNeohDHFRPA-Z/TM, pcDNA3.1(+)dNeohEF-1αPA-Z/TM,pcDNA3.1(+)dNeohUbCPA-Z/TM, pcDNA3.1(+)dNeohICIPA-Z/TM, pcDNA3.1(+)-Z/TM(FIG. 4) and pcDNA3.1(+)dNeo-Z/TM, respectively(pcDNA3.1(+)mPGKPA-MBP/TM, pcDNA3.1(+)hPGKPA-MBP/TM,pcDNA3.1(+)hDHFRPA-MBP/TM, pcDNA3.1(+)hEF-1αPA-MBP/TM,pcDNA3.1(+)hUbCPA-MBP/TM, pcDNA3.1(+)hICIPA-MBP/TM, as well aspcDNA3.1(+)dNeomPGKPA-MBP/TM, pcDNA3.1(+)dNeohPGKPA-MBP/TM,pcDNA3.1(+)dNeohDHFRPA-MBP/TM, pcDNA3.1(+)dNeohEF-1αPA-MBP/TM,pcDNA3.1(+)dNeohUbCPA-MBP/TM, pcDNA3.1(+)dNeohICIPA-MBP/TM,pcDNA3.1(+)-MBP/TM and pcDNA3.1(+)dNeo-MBP/TM.

The correctness of the constructs described in the example 1 to 3 wasconfirmed by sequencing.

The expression vectors described in the examples 1 to 3 are used in themethods according to the invention for the search for T-cell epitopesand for the detection of T-cells with known epitope-restriction,respectively, where the measurable change of the expression of apolypeptide that is naturally expressed in the APC as a result of anepitope-specific recognition by means of a T-cell, serves as a marker,for example the naturally increased expression of the OX40 ligand or ofthe 4-1 BB ligand.

EXAMPLE 4

Cloning of gene transfer vectors comprising a first promoter which isspecifically inducible in antigen presenting cells by theepitope-specific contact with a T-cell, comprising a nucleic acid beingfunctionally linked to this first promoter and encoding a marker gene,further comprising a second promoter being constitutive in antigenpresenting cells and comprising a nucleic acid being functionally linkedto this second promoter.

Cloning of the Vector Backbone pcDNA3.1(+)OX40LAeGFP According to theInvention as well as the Vectors pcDNA3.1(+)OX40LAeGFP-Z andpcDNA3.1(+)OX40LAeGFP-MBP According to the Invention

The starting vector for the cloning of the vector backbonespcDNA3.1(+)OX40LAeGFP according to the invention (FIG. 5A), as well asfor the vectors pcDNA3.1(+)OX40LAeGFP-Z (FIG. 5B) andpcDNA3.1(+)OX40LAeGFP-MBP (FIG. 5C) according to the invention, was theplasmid pcDNA3.1(+) (Invitrogen). The promoter of the OX40 ligand whichis inducible by an epitope-specific contact of a T-cell with anepitope-presenting APC was amplified from total DNA from human PBMC bymeans of the PCR method.

The 979 bp comprising sequence of the promoter of the OX40 ligand wasgenerated by PCR using the following amplification conditions:introductory denaturation step: 95° C., 2 min., followed by twosequential 3-step PCRs with the following conditions: first round ofPCR-amplification (10 cycles): denaturation: 95° C., 45 sec.; annealing50° C., 1 min.; elongation: 72° C., 2 min. Subsequently a second roundof PCR-amplification followed (30 cycles) with the following conditions:denaturation: 95° C., 45 sec.; annealing 60° C., 1 min.; elongation: 72°C., 2 min. with a final polymerisation step at 72° C. for 10 min. withsubsequent permanent cooling at 4° C. For amplification the primers P35(5′ primer: 5′-GGCGGGGGATCCGGTACCTGGTGTCTATTG-3′; SEQ ID NO: 40) and P36(3′ primer: 5′-GGCGGGAGATCTCTTCACAATCTGGGTAG-3′; SEQ ID NO: 41) wereused. The restriction sites contained within the primers (BamHI in the5′ primer; BglII in the 3′ primer) are depicted in italics. The sequenceof the amplified PCR fragment is depicted in FIG. 5 and SEQ ID NO: 1.The obtained PCR product was purified and digested with the restrictionenzymes BamHI and BglII, purified and cloned into the BglII-linearisedplasmid pcDNA3.1(+). The plasmid generated in such a way comprising 641bp was denoted pcDNA3.1(+)OX40L.

Subsequently the 224 bp comprising bovine growth hormone (BGH)polyadenylation sequence was amplified from the pcDNA3.1(+) vector byPCR using suitable PCR conditions (introductory denaturation step: 95°C., 2 min., subsequent 3-step PCR (35 cycles) with the followingconditions: denaturation: 95° C., 30 sec.; annealing 55° C., 1 min.;elongation: 72° C., 1 min. and a final polymerisation step at 72° C. for10 min. with subsequent permanent cooling at 4° C.). For amplificationthe primers P37 (5′ primer: 5′-GGCGGGAGATCCTGTGCCTTCTAGTTGCC-3′; SEQ IDNO: 42) and P38 (3′ primer: 5′-GGCGGGGGATCCCCATAGAGCCCACCGC-3′; SEQ IDNO: 43) were used. The restriction sites (BglII in the 5′ primer; BamHIin the 3′ primer) contained within the primers are depicted in italics.The PCR-amplified band was purified as described previously, restrictedwith the restriction enzymes BglII and BamHI, and the in such a wayobtained band was ligated into the BglII-linearised plasmidpcDNA3.1(+)0X40L. The vector generated in such a way was denotedpcDNA3.1 (+)OX40LA.

Subsequently the gene for the “enhanced green fluorescent protein”(eGFP) was amplified from the plasmids pRC/CMV-EGFP (Vogel et al., 1998,BioTechniques 143, 1967-1983) by PCR using suitable PCR conditions(introductory denaturation step: 95° C., 2 min., subsequently a 3-stepPCR (35 cycles) with the following conditions: denaturation: 95° C., 30sec.; annealing 55° C., 1 min.; elongation: 72° C., 2 min., and a finalpolymerisation step at 72° C. for 10 min. with subsequent permanentcooling at 4° C.). For amplification the primers P39 (5′ primer:5′-GGCGGGGGATCCCGCCACCATGGTGAGCAAGG-3′; SEQ ID NO: 44) and P40 (3′primer: 5′-GGCGGGAGATCTTTACTTGTACAGCTCGTCC-3′; SEQ ID NO: 45) were used.The PCR-amplified specific band with a size of 752 bp was purified asdescribed previously, digested with the restriction enzymes BamHI andBglII, and the obtained band was ligated into the BglII-linearisedplasmid pcDNA3.1(+)OX40LA. The in such a way generated vector comprising7378 bp was denoted pcDNA3.1(+)OX40LAeGFP (FIG. 5A). This vector can beused as the vector backbone for the cloning of the vectors according tothe invention for the search for epitopes and the detection of reactiveT-cells.

Subsequently, the cDNA for the EBV BZLF-1 protein was amplified fromviral DNA of the EBV strain B95-8 within the total DNA of infectedmarmoset cells by PCR using suitable PCR-conditions (introductorydenaturation step: 95° C., 2 min., subsequently a 3-step PCR (15 cycles)with the following conditions: denaturation: 95° C., 30 sec.; annealing52° C., 1 min.; elongation: 72° C., 2min., followed by 25 cycles withthe following conditions: denaturation: 95° C., 30 sec.; annealing 52°C., 1 min.; elongation: 72° C., 2 min., and a final polymerisation stepat 72° C. for 10 min. with a subsequent permanent cooling at 4° C.). Inthe case of the PCR reaction for the amplification of the BZLF-1 cDNA2.5% DMSO were added to the PCR sample. For amplification the primersP41 (5′ primer: 5′-GGCGGTCTAGATTAGAAATTTAAGAGATCC-3′; SEQ ID NO: 46) andP42 (3′ primer: 5′-GGCGGGGAATTCATGATGGACCCAAACTCG-3′; SEQ ID NO: 47)were used. The PCR-amplified 970 nucleotide comprising band was purifiedas described previously, restricted with the restriction enzymes XbaIand EcoRI, and the obtained band was ligated into the respectively withEcoRI and XbaI linearised plasmid pcDNA3.1(+)OX40LAeGFP. The vectorgenerated in such a way was denoted pcDNA3.1(+)OX40LAeGFP-Z (FIG. 5B).

Alternatively, the cDNA for the MBP protein was amplified from total DNAfrom brain cells by PCR using suitable conditions (introductorydenaturation step: 95° C., 2 min.; subsequently followed by a 3-step PCR(35 cycles) with the following conditions: denaturation: 95° C., 30sec.; annealing 55° C., 1 min.; elongation: 72° C., 2 min. and a finalpolymerisation step at 72° C. for 10 min. with a subsequent permanentcooling at 4° C.). For amplification the primers P43 (5′ primer:5′-GGCGGTCTAGAATGGCGTCACAGAAGAGACC-3′; SEQ ID NO: 48) and P44 (3′primer: 5′-GGCGGGGAATTCTCAGCGTCTAGCCATGGG-3′; SEQ ID NO: 49) were used.The PCR-amplified 585 nucleotides comprising band was purified asdescribed previously, restricted with the restriction enzymes XbaI andEcoRI, and the obtained bands were ligated into the respectively withEcoRI and XbaI linearised plasmid pcDNA3.1(+)OX40LAeGFP. The vectorgenerated in such a way was denoted pcDNA3.1(+)OX40LAeGFP-MBP (FIG. 5C).

In addition, a first promoter (P₁), here exemplary the OX40 ligandpromoter and 4 variants of the 4-1 BB ligand promoter, which isspecifically inducible in antigen presenting cells by the epitopespecific contact with a T-cell, as well as a nucleic acid encoding amarker gene, here exemplary the eGFP, which is functionally linked tothis first promoter, were inserted in all expression plasmids which aredescribed in the examples 1-3.

For this, three nucleic acid modules, the sequence of a first promoter(P₁) being inducible in APC, a BGH polyadenylation sequence and asequence for a marker, exemplary the nucleic acid encoding eGFP, wereeach restricted and purified after performed PCR-amplification andinserted sequentially in three steps into a BglII-restricted expressionvector each. Hereby, for all cloning steps the BglII-restriction sitewas used which is localised at position 13 of the vector pcDNA3.1(+)(Invitrogen) that forms the basis for all generated plasmids. OtherBglII-restriction sites which were generated within the vectors due tothe performed cloning steps were deleted by means of a QuickChangeSite-Directed Mutagenis Kit following the manufacturer's (Stratagene)protocol in such a way that the amino acid sequence of the polypeptideand the functionality of the nucleic acid, respectively, staysunchanged. The selection of suited oligonucleotides for mutagenesis andthe performance of the method are established and state of the art.

Amplification of the Human OX40 Ligand Promoter

The 979 bp comprising sequence of the murine PGK promoter was obtainedby PCR from isolated chromosomal DNA from human PBMCs using thefollowing conditions: introductory denaturation step: 95° C., 2 min.,subsequently followed by two 3-step PCRs with the following conditions:first round of PCR-amplification (10 cycles): denaturation: 95° C., 45sec.; annealing 50° C., 1 min.; elongation: 72° C., 2 min. Subsequentlyfollowed by a second round of PCR-amplification (30 cycles) with thefollowing conditions: denaturation: 95° C., 45 sec.; annealing: 60° C.,1 min.; elongation: 72° C., 2 min. with a final polymerisation step at72° C. for 10 min. with subsequent permanent cooling at 4° C. Foramplification the primers P45 (5′ primer:5′-GGCGGGGGATCCGGTACCTGGTGTCTATTG-3′; SEQ ID NO: 50) and P46 (3′ primer:5′-GGCGGGAGATCTCTTCACAATCTGGGTAG-3′; SEQ ID NO: 51) were used. Therestriction sites (BamHI in the 5′ primer; BglII in the 3′ primer)contained within the primers are depicted in italics.

Amplification of the Human 4-1 BB Ligand Promoter (Long Variant of the4-1 BB Ligand Promoter (VI))

The 2047 bp comprising sequence of the human 4-1 BB ligand promoter wasisolated by PCR from isolated chromosomal DNA from human PBMCs using thefollowing amplification conditions: introductory denaturation step: 98°C., 2 min., followed by three subsequent 3-step PCRs with the followingconditions: first round of PCR-amplification (30 cycles): denaturation:95° C., 1 min.; annealing: 66.1° C., 1 min.; elongation: 72° C., 4.5min. Subsequently followed by a second round of PCR-amplification (15cycles) with the following conditions: denaturation: 98° C., 1 min.;annealing: 58° C., 1 min.; elongation: 72° C., 4.5 min. and a thirdround of PCR-amplification (25 cycles) with the following conditions:denaturation: 98° C., 1 min.; annealing: 71° C., 1 min.; elongation: 72°C., 4.5 min. with a final polymerisation step at 72° C. for 10 min. withsubsequent permanent cooling at 4° C. For amplification the primers P47(5′ primer: 5′-GGCGGGGGATCCCCGGATTGGCCGCCTCCAGCAG-3′; SEQ ID NO: 52) andP48 (3′primer: 5′-GGCGGGGGATCCGACGAGAGACTGCGGGAAGACAC-3′; SEQ ID NO: 53)were used. The obtained PCR-amplificate was subsequently purified,restricted with BamHI and inserted in the respectively BamHI-restrictedpcDNA3.1(+)-vector. The obtained construct was denotedpcDNA3.1(+)4-1BBL. Subsequently, the BglII-restriction site within the4-1 BB ligand promoter (at position 1625 to 1630 of the 2023 nucleotidecomprising full length promoter) was deleted by means of a QuickChangeSite-Directed Mutagenesis Kit following the manufacturer's (Stratagene)protocol by the exchange of a nucleotide at the position 1626 (1626 G toA)-(1624-AGATCT-1631) to 1626 (1624-AAATCT-1631). The resulting plasmidobtained the denotation pcDNA3.1(+)4-1BBL1626.

Amplification of the Mutated (1626 G to A) (Human 4-1 BB Ligand Promoter(Long Variant of the 4-1 BB Ligand Promoter (V1))

The 2047 bp comprising sequence of the human 4-1 BB ligand promoter wasisolated by PCR from the plasmid pcDNA3.1(+)4-1BBL1626 using thefollowing amplification conditions: introductory denaturation step: 98°C., 2 min., subsequently followed by three sequential 3-step PCRs withthe following conditions: first round of PCR-amplification (30 cycles):denaturation: 95° C., 1 min.; annealing: 66.1° C., 1 min.; elongation:72° C., 4.5 min. Subsequently followed by a second round ofPCR-amplification (15 cycles) with the following conditions:denaturation: 98° C., 1 min.; annealing: 58° C., 1 min.; elongation: 72°C., 4.5 min., and a third round of PCR-amplification (25 cycles) withthe following conditions: denaturation: 98° C., 1 min.; annealing: 71°C., 1 min.; elongation: 72° C., 4.5 min. with a final polymerisationstep at 72° C. for 10 min. with a subsequent permanent cooling at 4° C.For amplification the primers P49 (5′ primer:5′-GGCGGGGGATCCCCGGATTGGCCGCCTCCAGCAG-3′; SEQ ID NO: 54) and P50 (3′primer: 5′-GGCGGGAGATCTGACGAGAGACTGCGGGAAGACAC-3′; SEQ ID NO: 55) wereused.

Amplification of the Human 4-1 BB Ligand Promoter (in the 5′ RegionTruncated Variant of the 4-1 BB Ligand Promoter (V2))

The 1433 bp comprising sequence of the human 4-1 BB ligand promoter (V2)was isolated by PCR from the plasmid pcDNA3.1(+)4-1BBL1626 using thefollowing amplification conditions: introductory denaturation step: 98°C., 2 min., subsequently followed by three sequential 3-step PCRs withthe following conditions: first round of PCR-amplification (30 cycles):denaturation: 95° C., 1 min.; annealing: 64.1° C., 1 min.; elongation:72° C., 4.5 min. Subsequently followed by a second round ofPCR-amplification (15 cycles) with the following conditions:denaturation: 98° C., 1 min.; annealing: 58° C., 1 min.; elongation: 72°C., 3.5 min., and a third round of PCR-amplification (25 cycles) withthe following conditions: denaturation: 98° C., 1 min.; annealing: 69°C., 1 min.; elongation: 72° C., 3.5 min. with a final polymerisationstep at 72° C. for 10 min. with subsequent permanent cooling at 4° C.For amplification the primers P51 (5′ primer:5′-GGCGGGGGATCCGGAGCCAGAGATAGGGAGAGTC-3′; SEQ ID NO: 56) and P50 (3′primer: 5′-GGCGGGAGATCTGACGAGAGACTGCGGGAAGACAC-3′; SEQ ID NO: 55) wereused.

Amplification of the Human 4-1 BB Ligand Promoter (in the 5′ RegionFurther Truncated Variant of the 4-1 BB Ligand Promoter (V3))

The 874 bp comprising sequence of the human 4-1 BB ligand promoter (V3)was isolated by PCR from the plasmid pcDNA3.1(+)4-1BBL1626 using thefollowing amplification conditions: introductory denaturation step: 98°C., 2 min., subsequently followed by three sequential 3-step PCRs withthe following conditions: first round of PCR-amplification (30 cycles):denaturation: 95° C., 1 min.; annealing: 66° C., 1 min.; elongation: 72°C., 2 min. Subsequently followed by a second round of PCR-amplification(15 cycles) with the following conditions: denaturation: 98° C., 1 min.;annealing: 55° C., 1 min.; elongation: 72° C., 2 min., and a third roundof PCR-amplification (25 cycles) with the following conditions:denaturation: 98° C., 1 min.; annealing: 69° C., 1 min.; elongation: 72°C., 2 min. with a final polymerisation step at 72° C. for 10 min. withsubsequent permanent cooling at 4° C. For amplification the primers P52(5′ primer: 5′-GGCGGGGGATCCCAGCCAGAGACAGCGACAGAG-3′; SEQ ID NO: 57) andP50 (3′ primer: 5′-GGCGGGAGATCTGACGAGAGACTGCGGGAAGACAC-3′; SEQ ID NO:55) were used.

Amplification of the Human 4-1 BB Ligand Promoter (in the 5′ RegionFurther Truncated Variant of the 4-1 BB Ligand Promoter (V4))

The 386 bp comprising sequence of the human 4-1 BB ligand promoter (V4)was isolated by PCR from the plasmid pcDNA3.1(+)4-1BBL1626 using thefollowing amplification conditions: introductory denaturation step: 98°C., 2 min., subsequently followed by three sequential 3-step PCRs withthe following conditions: first round of PCR-amplification (30 cycles):denaturation: 95° C., 1 min.; annealing: 66° C., 1 min.; elongation: 72°C., 2 min. Subsequently followed by a second round of PCR-amplification(15 cycles) with the following conditions: denaturation: 98° C., 1 min.;annealing: 55° C., 1 min.; elongation: 72° C., 2 min., and a third roundof PCR-amplification (25 cycles) with the following conditions:denaturation: 98° C., 1 min.; annealing: 69° C., 1 min.; elongation: 72°C., 2 min. with a final polymerisation step at 72° C. for 10 min. withsubsequent permanent cooling at 4° C. For amplification the primers P53(5′ primer: 5′-GGCGGGGGATCCCCCACTGCAGAGGCAATCAACAAG-3′; SEQ ID NO: 58)and P50 (3′ primer: 5′-GGCGGGAGATCTGACGAGAGACTGCGGGAAGACAC-3′; SEQ IDNO: 55) were used.

The 224 bp comprising bovine growth hormone (BGH) polyadenylationsequence and the gene for the “enhanced green fluorescent protein”(eGFP) were obtained by PCR as already described in example 4.

The three nucleic acid components for the expression of a marker bymeans of an inducible promoter (P₁), namely the nucleic acid sequence ofa promoter, of a BGH polyadenylation signal sequence and of a nucleicacid encoding a marker were—as described in the following—sequentiallyinserted into the plasmids listed hereafter. Step 1: digestion of thenucleic acid of the promoter obtained by PCR with the enzymes BamHI andBglII and ligation of the thereby obtained nucleic acid into BglIIrestricted target vectors. Step 2: digestion of the obtained nucleicacid of the BGH polyadenylation signal sequence obtained by PCR with theenzymes BglII and BamHI and ligation of the thereby obtained nucleicacid into the target vectors generated in step 1 and again restrictedwith BglII. Step 3: digestion of the nucleic acid of the marker (eGFP)obtained by PCR with the enzymes BamHI and BglII and ligation of thethereby obtained nucleic acid into the target vectors generated in step2 and again restricted with BglII.

With this method 4 different first promoters, the OX40L, the 4.1BBL(V1), the 4.1 BBL(V2), the 4.1 BBL(V3) and the 4.1 BBL(V4) promoter,being specifically inducible in antigen presenting cells by theepitope-specific contact with a T-cell as well as a nucleic acidencoding a marker gene (eGFP) being functionally linked with this firstpromoter were inserted into the plasmids pcDNA3.1(+), pcDNA3.1(+)dNeo,as well as into the expression vectors listed in the examples 1 to 3,containing a second promoter being constitutive in antigen-presentingcells and containing a nucleic acid being functionally linked to thissecond promoter. The plasmids which were generated in such a way weredenoted by the addition of the denotation of the promoter (OX40L,4.1BBL(V1), 4.1BBL(V2), 4.1BBL(V3) or 4.1BBL(V4)) to the original name.So for example the plasmid pcDNA3.1(+)-Z/LAMP is denotedpcDNA3.1(+)-Z/LAMP/OX40L (FIG. 5D) after insertion of the nucleic acidfor the OX40 ligand promoter, the BGH polyadenylation signal sequenceand the marker eGFP.

Potential restriction endonuclease restriction sites which mightinterfere with the procedure of the cloning steps described in theexamples 1 to 4 were deleted by means of a QuickChange Site-DirectedMutagenesis Kit following the manufacturer's (Stratagene) protocol insuch a way that the amino acid sequence of the polypeptide and thefunctionality of the nucleic acid, respectively, stays unchanged. Theselection of suitable oligonucleotides for mutagenesis and theperformance of the method are established and state of the art. Thecorrectness of the sequence and the orientation of the polynucleotideswhich were integrated into the respective vectors were confirmed bysequencing.

EXAMPLE 5 Transfection of the Vectors According to the Invention intoPopulations of Purified APC or Purified PBMC by Means of theNucleovector™ Technology (Amaxa)

The preparation of peripheral mono-nucleated cells of the blood (PBMC)from whole blood of voluntary donors or patients was performed asdescribed hereafter: freshly extracted whole blood (50 to 100 ml; notolder than 8 h) of a healthy test person was spiked with 20 IU/mlheparin. 50 ml-Leukosep-vials with a separating membrane were filledwith 15 ml Ficoll-Histopaque (Sigma, Deisenhofen, Germany) andcentrifuged for 2 min. at 500×g. Subsequently, the tubes were filledwith the heparinised whole blood and mixed 1:1 (vol/vol) with sterilePBS/0.5% BSA. The samples were centrifuged for 30 to 40 min. at 400×gwithout decelerating the rotor at the end of the spin. Thelymphocyte-containing turbid interphase was removed carefully and washedin 3 volumes of T-cell medium (RPMI 1640 medium with 10% heatinactivated (30 min. at 56° C.) human AB serum, 2 mM glutamine and 100mg/ml kanamycin and gentamycin, respectively, (all components byPanSystem, Aidenbach, Germany)) for three times (200×g; 10 to 15 min. atroom temperature) and resuspended with a cell number of 2×10⁶ cells/mlin RPMI 1640 medium (Gibco) with 10% human AB serum, 100 μg/mlstreptomycin, 100 U/ml penicillin and 2 mM GlutaMAX (Invitrogen/LiveTechnologies) and stored in a humidified incubator with 5% CO₂ gassingat 37° C. prior to the further purification of defined APC- and T-cellpopulations. By means of this method from 100 ml whole blood (60%erythrocytes; 40% leukocytes) are extracted about 1×10⁷ to 1×10⁸ PBMCswith an average composition of about 60%-70% T-cells, 5%-10% B-cells,10%-20% monocytes/macrophages and about 10% of other cells.

The purification of B-cells from freshly prepared PBMCs was performed bya positive selection by means of immuno-magnetic CD19 MicroBeatsfollowing the manufacturer's protocol (Miltenyi Biotec).

For this, the isolated PBMC were counted and resuspended in aconcentration of 10⁷ cells/80 μl MACS buffer (PBS, 0.5% BSA and 2 mMEDTA, degassed). For each 10⁷ PBMC 20 μl MACS CD10 MicroBeats were addedand incubated for 15 min. at 6° C. Subsequently, the labelled cellsuspension was mixed with 2 ml buffer for 10⁷ PBMC each and centrifugedfor 10 min. at 300×g in a Hettich-Rotanta table centrifuge. Thesupernatant was removed carefully and the cells were resuspended in 500μl MACS-buffer. Prior to loading on a column, the cells wereindividualised by filtration through a cell sieve, 70 μm mesh size(BectonDickinson, Heidelberg, Germany). For the positive selection ofthe magnetically labelled cells, columns of the type XS+ were chosen(max. 10⁹ positive cells) and placed into a MACS separator (MiltenyiBiotec, Germany). The columns were washed once to twice with 10 ml MACSbuffer until the flowthrough was clear. Subsequently, the individualisedcells were loaded on the prepared column and the negative fraction waseluted. The column was again washed 3× with MACS buffer, withdrawn fromthe MACS-separator and transferred into a sterile plastic vial. TheB-cells were subsequently eluted by the loading of MACS buffer and theexertion of a feeble pressure by a plunger belonging to the column.After this, the cells were stored until transfection with a cell numberof 1×10⁶ cells/ml in RPMI 1640 medium (Gibco) with 10% human AB serum,100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mM GlutaMAX(Invitrogen/Live Technologies) at 37° C. in a humidified incubator with5% CO₂ gassing.

In the same manner CD3⁺ T-cells were depleted from PBMCs by positiveselection using MACS CD3 MicroBeads and the obtained cell suspension wascharacterised in regard of its composition by means of FACS before thetransfer of nucleic acid.

Subsequently, the obtained cell populations were characterised in regardto their composition by means of a FACS device using suitable,dyestuff-labelled antibodies against cell type-specific surfacemolecules (CDs). By using this method, B-cells could be obtained havinga purity of about 83% (see table 1A). Analogously, CD3-positive T-cellswere depleted from PBMCs by means of the Miltenyi method following themanufacturer's protocol and the resulting cell populations werecharacterised by means of FACS analysis. The obtained cell suspensionwas composed of 3.37% B-cells (CD19⁺), 58% CD4⁺-cells (monocytes andremaining T-cells), about 1.55% contaminating T-cells (CD3⁺); thereofabout 0.35% T-helper cells and 1.2% CTL (CD8⁺) and 58% monocytes (CD3⁻,CD4⁺).

Transfection of CD3⁺ Depleted PBMC and Purified B-Cells, Respectively,with the Vectors According to the Invention.

Exemplarily, the transfection of CD3⁺ depleted PBMC (FIG. 6A) and thepurified B-cells (FIG. 6B), respectively, was performed with the vectorpcDNA3.1(+)dNeo-eGFP according to the invention by means of theNucleovector™ technology using the human B cell Nucleovector™ kitfollowing the detailed manufacturer's protocol (Amaxa, Germay). Ascontrols the cells were treated with the plasmid pcDNA3.1(+). At this1×10⁷ to 3×10⁷ CD3 depleted PBMCs and purified CD19⁺ B-cells,respectively, per ml were centrifuged (200×g, 10 min. at roomtemperature) and the sedimented cells were resuspended in the “HumanB-Cell Nucleofactor” solution at a concentration of 1×10⁶ to 3×10⁶cells/ml. After this 100 μl of the cell suspension was spiked with 5 μlDNA and the sample was pipetted into a cuvette avoiding air bubbles.Subsequently, the cells were transfected by means of a Nucleofectordevice using a preset program of the manufacturer (Amaxa, Program U-15).After performed transfection 500 μl of pre-warmed medium (RPMI 1640medium (Gibco) with 10% human AB serum, 100 μg/ml streptomycin, 100 U/mlpenicillin and 2 mM GlutaMAX (Invitrogen/Live Technologies)) were addedand the cells were incubated afterwards at 37° C. in a humidifiedincubator with 5% CO₂ gassing. The transfection efficiency wasdetermined at several time points after performed transfection (forexample 12 to 72 hours, exemplary for 17 hours (FIGS. 6A, B; tables2A-D)) by means of a FACS device measuring the number of fluorescentcells (detection of a GFP reporter). For this, the transfected cellswere 2× washed with FACS buffer (PBS without bivalent ions, 1% FCS, 0.9mg/ml acid) and after this resuspended in a density of 5×10⁵ cells inFACS buffer. Subsequently, the number of fluorescent cells wasdetermined by means of a FACS device (FACS Calibur, BD) by measuring ofthe fluorescence 1 (FL1) against the forward scatter. These experimentsshowed that due to the Nucleovector™ Technology (Amaxa) about 13.5% ofB-cells and 30% of the monocytes in the mixed population of CD3 depletedPBMCs show a significant eGFP production (table 2A). In populations ofhigh-purity B-cells the transfection rate of B-cells amounted to onlyabout 7.4% in contrast (table 2B). The cells being transfected with acontrol vector pcDNA3.1(+) did not show any significant eGFP productionin these experiments (tables 2C,D). These analyses substantiate thesuitability of the Nucleovector™ Technology of Amaxa for the transfer ofthe vectors according to the invention into APC. TABLE 1A FACS analysisof the composition of the population of CD3⁺ depleted PBMCs which wasobtained by the Miltenyi method for CD3 depletion. cell type % CD19⁺3.37 CD4⁺ 58.39 CD3⁺ 1.52 CD3⁻CD4⁺ 58.30 CD3⁺CD4⁺ 0.37 CD3⁺CD8⁺ 1.19

TABLE 2C Detection of the fluorescence from different cell populationsin CD3 depleted PBMCs by means of FACS analysis of the nucleofectionwith pcDNA3.1(+) (FIG. 6A, middle) fluorescent cells % total 0.28 CD19⁺0.70 CD4⁺/Th 1.03 CD3⁺ 0.88 CD3⁺CD4⁻ 0.27

TABLE 2A Detection of the fluorescence from different cell populationsin CD3 depleted PBMCs by means of FACS analysis after nucleofection withpcDNA3.1dNeo-eGFP (FIG. 6A, right) fluorescent cells % total 34.94 CD19⁺13.48 CD4⁺/Th 3.79 CD3⁺ 5.01 CD3⁺CD4⁻ 30.06

TABLE 1B FACS-analysis of the composition of the B-cells which wereobtained by means of the Miltenyi method for B-cell separation cell type% CD19⁺ 83.38 CD4⁺ 0.96 CD3⁺ 5.77 CD3⁺CD4⁺ 1.79 CD3⁺CD8⁺ 1.13

TABLE 2D Detection of the fluorescence in enriched non-purified B-cellsby FACS after nucleofection with pcDNA3.1(+) (FIG. 6B, middle)fluorescent cells % total 0.31 CD19⁺ 0.43

TABLE 2B Detection of the fluorescence in enriched non-purified B-cellsby FACS after nucleofection with pcDNA3.1dNeo-eGFP (FIG. 6B, right)fluorescent cells % total 8.92 CD19⁺ 7.39

In contrast to this, transfection rates of more than 50% could beachieved by means of the Femtosecond Laser Technology in the case ofdifferent purified APC populations (monocytes, B-cells, dendriticcells). For this, 1-5×10⁵ APC were suspended each in a miniaturisedsterile cell chamber in 0.5 ml culture medium and 0.2 μg of thepcDNA3.1(+)dNeo-eGFP plasmid. Subsequently, the cells were treatedaccording to an available protocol (Tirlapur and König, (2001) Nature,418, 290) and the transfection efficiency was determined as alreadydescribed by means of a FACS device. This method is presently beingdeveloped by the company Jenlab GmbH to series-production readiness butis at present not yet commercially available.

EXAMPLE 6 Enhancement of the Marker Expression in the APC According tothe Invention After Contact with Epitope-Specific T-Cells

The testing for suitability of the APC according to the invention forthe detection of epitope-specific T-cells and for the search forepitopes was performed on the basis of a very well characterised modelsystem. This system is based on the experimental results that all HLA-B8positive, EBV positive test persons exhibit CD8⁺ cytotoxic T-cells witha high precursor frequency which recognise a specific epitope (RAKFKQLL;amino acid 190-197) within the EBV protein BZLF-1 (Bogedain et al.,1995, J. Virol. 69, 4872-4879; Benninger-Döring et al., 1999, Virol.264, 289-297).

A: Enhanced Expression of a Natural Marker (OX40L) on Transfected APCAfter Incubation with the Plasmid pcDNA3.1(+)dNeo-Z/LAMP According tothe Invention

In the case of these examinations, monocytes of an HLA-B8 positive EBVsero-positive donor (SD) and as a control of an HLA-B8 positive, EBVsero-negative donor (SN) were negatively purified using a mixture ofantibody-labelled magnetic beads (Miltenyi) according to themanufacturer's protocol, and the purified monocytes were differentiatedin RPMI medium (10% FCS) by the addition of 25 ng/ml IL-4 and 800 U/mlGM-CSF to dendritic cells (DC). At this, the medium was changed on day 4of the incubation and exchanged by a medium containing GM-CSF, IL-4 and10 ng/ml TNF-α. After culturing for 7 days the cells were transfectedwith the plasmid pcDNA3.1 (+)dNeo-Z/LAMP and as a control with theplasmid pcDNA3.1(+)dNeo using the Nucleofactor or Femtolaser technology.After further culturing for 12 to 36 hours in RPMI 1640 medium (Gibco)with 10% human AB serum, 100 μg/ml streptomycin, 100 U/ml penicillin and2 mM GlutaMAX (Invitrogen/Live Technologies) the cells subsequently wereincubated in 20 parallel approaches in different ratios (10:1 to 1:10)of DC and T-cells which were modified according to the invention andwere derived from the same patients, with a whole cell number of 5×10⁶cells in RPMI 1640 medium (Gibco) with 10% human AB serum, 100 μg/mlstreptomycin, 100 U/ml penicillin and 2 mM GlutaMAX (Invitrogen/LiveTechnologies) for 2 to 72 hours at 37° C. in a humidified incubator with5% CO₂ gassing. After this, the expression of the OX40 ligand on thesurface of the dendritic cells was determined by means of a FACSanalysis using the OX40L-specific antibody 5A8 (Imura et al. (1996), J.Exp. Med. 183:2185). Thereby in the case of about 2 to 20% (dependent onthe experimental protocol) of all pcDNA3.1(+)dNeo-Z/LAMP-plasmid treatedDC of the HLA-B8 positive EBV sero-positive donor (SD) in comparison tothe with the same plasmid treated DC of the HLA-B8 positive EBVsero-negative donor (SN) as well as with the pcDNA3.1(+)dNeo-controlvector treated DCs of both donors, a measurably increased surfaceexpression of the OX40L could be observed. This increased surfaceexpression of OX40L was measurable during a time period of 4 to 72hours. Similar results were obtained in co-cultivation experiments usingB-cells and monocytes as APC.

B: Enhanced Expression of a Natural Marker (OX40L) on Transfected APCAfter Transfection with the Plasmids pcDNA3(+)OX40LAeGFP-Z andpcDNA3.1(+)dNeo-Z/LAMP/OX40L According to the Invention

In the case of these analyses, B-cells as well as monocytes of an HLA-B8positive EBV sero-positive donor (SD) and as control of an HLA-B8positive EBV sero-negative donor (SN) were positively purified usingspecific antibody labelled magnetic beads (Miltenyi) following themanufacturer's protocol and were transfected with the plasmidspcDNA3(+)OX40LAeGFP-Z and pcDNA3.1(+)dNeo-Z/LAMP/OX40L and as a controlwith the vector pcDNA3.1(+)dNeo after a cultivation for 1 to 24 hoursusing the Nucleofector or Femtolaser technology. After a furthercultivation for 12-36 hours in RPMI 1640 medium (10% human AB serum, 100μg/ml streptomycin, 100 U/ml penicillin and 2 mM GlutaMAX) the cellswere subsequently incubated in 20 parallel approaches in differentratios (10:1 to 1:10) of APC (B-cells or monocytes) and T-cells whichwere modified according to the invention and derived from the samepatients, with a total cell number of 5×10⁶ cells in RPMI 1640 medium(10% human AB serum, 100 μg/ml streptomycin, 100 U/ml penicillin and 2mM GlutaMAX) for 12 to 72 hours at 37° C. in a humidified incubator with5% CO₂ gassing. After this, the number of eGFP marker-positive APC wasdetermined by means of a FACS device according to the method describedin example 5. Hereby, in about 1 to 20% (dependent on the experimentalprocedure) of the pcDNA3(+)OX40LAeGFP-Z- andpcDNA3.1(+)dNeo-Z/LAMP/OX40L-plasmid transfected APC of the HLA-B8positive EBV sero-positive donor (SD) in comparison to the with the sameplasmid treated DC of the HLA-B8 positive EBV sero-negative donor (SN)as well as with the pcDNA3.1(+)dNeo control vector treated DCs of bothdonors, a measurable increased marker production could be observed. Thisincreased fluorescence was measurable over a time period of 4 to 72hours. Similar results were achieved using DC as APC.

EXAMPLE 7 Isolation of Marker-Positive APC by Means of the FACS-SortingMethod and Isolation of Polynucleotides from Selected Cells

For the identification of the polypeptides being recognised by thereactive T-cells of the HLA-B8-positive donor (SD) or APC, which showedan increased marker expression, were selected by means of the FACSsorttechnology, collected, and the total DNA was prepared from these APC.For transformation of the total DNA into bacteria 20 μl of a DH10Bbacterial suspension was mixed with the obtained total DNA andelectroporated for about 5 msec. at a voltage of 2400V. After this, thebacteria were shortly cooled on ice, and subsequently mixed with 1 ml LBmedium and incubated under shaking for 1.5 hours at 37° C. Finally,different amounts of the bacterial suspension were plated on LB_(AMP)selection plates. These were incubated overnight at 37° C. In theseexperiments a significant number of ampicillin resistant colonies/plate(10-100) could be generated by the transfection of 1 μg total DNA. ByPCR analysis of the bacterial clones using suited EBV BZLF-1-specificprimers as well as by sequencing of the plasmids obtained from thebacterial clones using suitable sequencing primers, it could be shownthat more than 90% of the resistant bacterial clones contained theexpression vectors encoding the BZLF-1 protein.

EXAMPLE 8 Detection of Specific Vector-Encoded Polynucleotides from theGenome of Stably Transfected Insect Cells

In the early 90ies a very sensitive method was described by Wolff andcolleges allowing to detect even episomal plasmids but also plasmidsbeing stably integrated into the genome of the transfected cells (Wolffet al., 1991). This detection method is based on the observation thatplasmids which are episomaly present inside the cell but not plasmidswhich have integrated stably into the genome of a host cell lead to theformation of antibiotics-resistant colonies after the extraction of thetotal DNA and a subsequent electroporation of the DNA into bacteria. Forthis the plasmid has to contain a bacterial origin of replication and aselectable bacterial resistance gene.

In control experiments a transformation efficiency of about 10⁸ coloniesper μg of transfected plasmid DNA could be determined afterelectroporation of 1 pg, 5 pg and 10 pg of the plasmid pAM-HBsAgencoding the small coat protein (HbsAg) of the hepatitis B virus (Demlet al., J. Virol. Methods (1999), 79:191-203). A comparableelectroporation efficiency was observed when the plasmid DNA was appliedtogether with 1 μg of chromosomal DNA from drosophila Schneider-2 (DS-2)cells. Therefore, the electroporation efficiency of plasmids is notaffected by the presence of chromosomal DNA. In contrast, byelectroporation of the respective linearised plasmid DNA only very viewampicillin-resistant bacterial colonies could be generated.

In order to test if the plasmid pAM-HbsAg, which has been transfectedinto DS-2 insect cells by lipofection, is present extra-chromosomal orintegrated into the genome of the cells, the total DNA was prepared fromthree independent samples of this cell line and transfected intobacteria. As a control, bacteria were transformed with total DNA fromnon-transfected DS-2 cells. In both cases only very viewampicillin-resistant colonies could be generated by the transfection of1 μg total DNA. By PCR analysis using HBsAg-specific primers it could beshown that none of these resistant bacterial clones contained the HBsAggene. Therefore, it can be deduced from these experiments that the totalDNA which was prepared from stably transfected DS-2 cells did notcontain any detectable amounts of episomaly present pAM-HBsAg DNA.

Further electroporation studies were to show that the plasmid DNA ispresent integrated into the genome of the stably transfected clonal DS-2cell line. For this purpose, total DNA was obtained from the stablytransfected DS-2 cell line and non transfected DS-2 cells, restrictedwith selected restriction enzymes, re-ligated and transfected intobacteria. By this procedure the integrated copies of the expressionplasmid pAM-HBsAg should be turned into extra-chromosomal plasmids whichcan be detected after electroporation into bacteria by facilitating anampicillin-resistance. The restriction enzyme EcoRV, which was used forthe restriction of the total DNA, singularly cuts the plasmid pAM-HBsAgin the region between the Mtn promoter and the reading frame for theHbsAg reporter gene. By digestion of the chromosomal DNA of the stablywith pAM-HBsAg transfected DS-2 cells with EcoRV, re-ligation andelectroporation into E. coli more than 200 ampicillin-resistant coloniescould be obtained. More than 80% of the established bacterial clonesproved HBsAg-positive in the PCR. For the further characterisation ofthese clones the plasmid DNA was prepared from E. coli and digested withEcoRV. Most of these bacterial clones contained the “input” plasmidpAM-HBsAg in the original size of about 8.2 kbp. These plasmids havingan unchanged size as compared to pAM-HBsAg, probably result fromplasmids that integrated in multiple copies to an integration spotwithin the genome of the host cell. About 15% of the analysedHBsAg-positive bacterial clones contained plasmids that showed a sizediffering from the co-expression plasmid pAM-HBsAg. These plasmids wereprobably excised from the genome at border regions of chromosomal DNAand the integrated plasmid DNA whereby besides the plasmid-encoded,another genomic EcoRV restriction site was used being located inproximity to the integrated plasmid. After transfection of bacteria withequally treated chromosomal DNA from non-transfected DS-2 cells 22ampicillin-resistant bacterial colonies could be generated. All of themproved HBsAg-negative in the PCR analysis.

Comparable results were obtained after electroporation of total DNA fromthe stably transfected DS-2 cell line and from non-transfected DS-2cells after digestion with the restriction endonuclease EcoRI. EcoRIcuts the plasmid pAM-HBsAg at two sites thereby producing two fragmentsof about 4.7 kbp and 3.5 kbp. Besides the coding region for themetallothionein (Mtn) promoter and the HBsAg-reporter construct thebigger fragment contains also the bacterial origin of replication andampicillin resistance and thus all important components for thegeneration of ampicillin-resistant bacteria after re-ligation andtransformation. In contrast, the smaller 3.5 kbp fragment contains nosequences which are necessary for the formation of ampicillin-resistantbacterial clones. After the re-ligation and electroporation of the DNAafter digestion with EcoRI 295 bacterial colonies could be counted onampicillin-containing LB-plates. By analysis of the purified plasmid DNAusing EcoRI restriction digest it could be shown that 43 of 46 testedHBsAg-positive clones contained a 4.7 kbp DNA-fragment. After an EcoRIdigest, 4 of the 46 extracted plasmids corresponded to the band patternof the EcoRI restricted pAM-HBsAg plasmid. After the analyticrestriction enzyme digest with EcoRI in the case of some plasmidpreparations smaller DNA fragments could be detected in addition to the4.7 kbp fragment. These fragments could be derived from chromosomalsequences which were cleaved out of chromosomal DNA during the EcoRIdigestion and re-ligated with the 4.7 kbp fragment to a bigger plasmid.About 5% of the analysed HBsAg-positive bacterial clones containexclusively 1 plasmid which includes the coding sequence of HBsAg butwhich differs in size from the 4.7 kbp fragment. These plasmids areprobably again derived from the border region between chromosomal DNAand the plasmid DNA which recombined in multiple copies at anintegration spot within the genome of the host cell. The generatedampicillin-resistant bacterial colonies which were generated aftertransfection of chromosomal DNA from non-transfected DS-2 cells provedHBsAg-negative in the PCR analysis with specific primers. These analysesshow that the described method is excellently suited for the isolationand characterisation of any nucleic acids being present episomal orintegrated into the genome of cells and therefore can also be used forthe isolation and characterisation of nucleic acids from marker-positivecells.

1. A gene transfer vector comprising a first promoter which isspecifically inducible in antigen presenting cells by epitope-specificcontact with a T-cell, a nucleic acid encoding a marker genefunctionally linked to said first promoter, a second promoter which isconstitutive in antigen presenting cells and a nucleic acid which isfunctionally linked to said second promoter.
 2. The gene transfer vectoraccording to claim 1, wherein said first promoter is a promoter for theOX40 ligand (OX40L), the 4-1 BB ligand (4-1 BBL), the co-stimulatoryprotein B7.1 (CD80), the co-stimulatory protein B7.2 (CD86) or for theFas ligand (FasL).
 3. The gene transfer vector according to claim 1,wherein said marker gene encodes a fluorescent polypeptide, luciferase(LUC), the alkaline phosphatase (AP), the secretory alkaline phosphatase(SEAP), the chloramphenicol acetyl transferase (CAT), thephotinus-luciferase, the β-glucoronidase (GUS), the renilla luciferase,the β-galactosidase (β-Gal), microbial structure or coat proteins orintracellular and membrane associated polypeptides that do not occurnaturally in APC.
 4. The gene transfer vector according to claim 1,wherein said second promoter is the early SV40 promoter, thecytomegaolvirus (CMV) promoter, the respiratory syncytial virus (RSV)promoter, the mouse mammary tumour virus (MMTV) LTR promoter, the humanimmune deficiency virus type 1 (HIV-1) LTR promoter, the adeno virusmajor late-promoter (Ad MLP), the herpes simplex virus (HSV) promoter,the murine 3-phosphoglycerate-kinase (PGK) promoter, the human PGK-1promoter, the human ubiquitin C-promoter, the human EF-1α promoter, thehuman β-casein promoter, the murine metallothionein promoter, the humanactin 5c promoter or the human ICI promoter.
 5. The gene transfer vectoraccording to claim 1, wherein said nucleic acid is derived from a cDNAlibrary or a genomic library.
 6. The gene transfer vector according toany claim 1, further comprising a bacterial origin of replication (ori)and a resistance gene.
 7. The gene transfer vector according to claim 6,further comprising recognition sequences for restriction endonucleasesflanking said second promoter, said nucleic acid being functionallylinked to said second promoter, and said bacterial origin of replicationand said coding sequence for the bacterial resistance.
 8. The genetransfer vector according to claim 1, further comprising a thirdpromoter functionally linked to a marker gene.
 9. A method for thedetection of epitope-specific T-cells comprising the following steps: a)isolating APC-containing and/or T-cell-containing body fluid, b)contacting and transducing the APC-containing body fluid with a genetransfer vector comprising gene transfer vector comprising a firstpromoter which is specifically inducible in antigen presenting cells byepitope-specific contact with a T-cell, a nucleic acid encoding a markergene functionally linked to said first promoter, a second promoter whichis constitutive in antigen presenting cells and a nucleic acid which isfunctionally linked to said second promoter, c) incubating the bodyfluid containing the transduced APC or isolated transduced APC with thebody fluid containing the T-cells or the isolated T-cells, and d)detecting marker-expressing APC.
 10. A method for the detection ofepitope-specific T-cells comprising the following steps: a) isolatingAPC-containing and/or T-cell-containing body fluid, b) contacting andtransducing APC-containing body fluid with a promoter (P₂) which isconstitutive in APC, and a nucleic acid which is functionally linked tosaid promoter, c) incubating the body fluid containing the transducedAPC or isolated transduced APC with the body fluid containing theT-cells or the isolated T-cells, and d) detecting marker-expressing APC.11. The method according to claim 9, wherein said APC-containing bodyfluid in step b) is blood, liquor, a purified PBMC-population or aseparated APC-population.
 12. The method according to claim 9, whereinsaid step of contacting takes 0.5 to 168 hours.
 13. The method accordingto claim 9, wherein said isolated T-cells are CD4⁺ T-cells, CD8⁺T-cells, CD4⁺ CD25⁺ regulatory T-cells, CD161⁺ NKT cells, or any mixtureof CD4⁺ T-cells, CD8⁺ T-cells, CD4⁺CD25⁺T-cells and CD161⁺ NKT-cells.14. The method according to claim 9, wherein said marker in step d) is apolypeptide which is naturally measurably induced or reduced in itsexpression after an epitope-specific recognition of an APC by a T-cell.15. An antigen presenting cell comprising a gene transfer vectorcomprising a first promoter which is specifically inducible in antigenpresenting cells by epitope-specific contact with a T-cell, a nucleicacid encoding a marker gene functionally linked to said first promoter,a second promoter which is constitutive in antigen presenting cells anda nucleic acid which is functionally linked to said second promoter. 16.The antigen presenting cell according to claim 15, wherein said antigenpresenting cell is a dendritic cell (Langerhans cell), a monocyte, amacrophage, a B-cell, a vascular endothelial cell, an epithelial or amesenchymal cell.
 17. (canceled)
 17. The method according to claim 9,wherein said step of contacting takes 0.5 to 2, 2 to 6, 6 to 12, 12 to36 or 36 to 168 hours.
 18. The method according to claim 10, whereinsaid step of contacting takes 0.5 to 168 hours.
 19. The method accordingto claim 18, wherein said step of contacting takes 0.5 to 2, 2 to 6, 6to 12, 12 to 36 or 36 to 168 hours.
 20. The method according to claim10, wherein said APC-containing body fluid in step b) is blood, liquor,a purified PBMC-population or a separated APC-population.
 21. The methodaccording to claim 10, wherein said isolated T-cells are CD4⁺ T-cells,CD8⁺ T-cells, CD4⁺CD25⁺ regulatory T-cells, CD161⁺ NKT cells, or anymixture of CD4⁺ T-cells, CD8⁺ T-cells, CD4⁺CD25⁺ T-cells and CD161⁺NKT-cells.
 22. The method according to claim 10, wherein said marker instep d) is a polypeptide which is naturally measurably induced orreduced in its expression after an epitope-specific recognition of anAPC by a T-cell.
 23. A method for the detection of target epitopes ofreactive T-cells comprising the following steps: a) isolatingAPC-containing and/or T-cell-containing body fluid, b) contacting andtransducing the APC-containing body fluid with a gene transfer vectorcomprising gene transfer vector comprising a first promoter which isspecifically inducible in antigen presenting cells by epitope-specificcontact with a T-cell, a nucleic acid encoding a marker genefunctionally linked to said first promoter, a second promoter which isconstitutive in antigen presenting cells and a nucleic acid which isfunctionally linked to said second promoter, c) incubating the bodyfluid containing the transduced APC or isolated transduced APC with thebody fluid containing the T-cells or the isolated T-cells, and d)detecting marker-expressing APC, and e) isolating the nucleic acid whichis functionally linked to said second promoter and which is encoding thetarget epitopes of reactive T-cells.
 24. The method of claim 23, furthercomprising characterizing of the nucleic acid which is functionallylinked to said second promoter and which is encoding the target epitopesof reactive T-cells.
 25. The method according to claim 23, wherein stepb) takes 0.5 to 168 hours.
 26. The method according to claim 23, whereinstep b) takes 0.5 to 2, 2 to 6, 6 to 12, 12 to 36 or 36 to 168 hours.27. The method according to claim 23, wherein said APC-containing bodyfluid in step b) is blood, liquor, a purified PBMC-population or aseparated APC-population.
 28. The method according to claim 23, whereinsaid isolated T-cells are CD4⁺ T-cells, CD8⁺ T-cells, CD4⁺CD25⁺regulatory T-cells, CD161⁺ NKT cells, or any mixture of CD4⁺ T-cells,CD8⁺ T-cells, CD4⁺CD25⁺ T-cells and CD161⁺ NKT-cells.
 29. The methodaccording to claim 23, wherein said marker in step d) is a polypeptidewhich is naturally measurably induced or reduced in its expression afteran epitope-specific recognition of an APC by a T-cell.
 30. A method forthe detection of target epitopes of reactive T-cells comprising thefollowing steps: a) isolating APC-containing and/or T-cell-containingbody fluid, b) contacting and transducing APC-containing body fluid witha promoter (P₂) which is constitutive in APC, and a nucleic acid whichis functionally linked to said promoter, c) incubating the body fluidcontaining the transduced APC or isolated transduced APC with the bodyfluid containing the T-cells or the isolated T-cells, and d) detectingmarker-expressing APC, and e) isolating the nucleic acid which isfunctionally linked to said second promoter and which is encoding thetarget epitopes of reactive T-cells.
 31. The method of claim 30, furthercomprising characterizing of the nucleic acid which is functionallylinked to said second promoter and which is encoding the target epitopesof reactive T-cells.
 32. The method according to claim 23, wherein stepb) takes 0.5 to 168 hours.
 33. The method according to claim 23, whereinstep b) takes 0.5 to 2, 2 to 6, 6 to 12, 12 to 36 or 36 to 168 hours.34. The method according to claim 23, wherein said APC-containing bodyfluid in step b) is blood, liquor, a purified PBMC-population or aseparated APC-population.
 35. The method according to claim 23, whereinsaid isolated T-cells are CD4⁺ T-cells, CD8⁺ T-cells, CD4⁺CD25⁺regulatory T-cells, CD161⁺ NKT cells, or any mixture of CD4⁺ T-cells,CD8⁺ T-cells, CD4⁺CD25⁺ T-cells and CD161⁺ NKT-cells.
 36. The methodaccording to claim 23, wherein said marker in step d) is a polypeptidewhich is naturally measurably induced or reduced in its expression afteran epitope-specific recognition of an APC by a T-cell.